- Email: [email protected]
DNA Damage Detection by Electrochemiluminescence Sensor of CdS Quantum Dots
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 41, Issue 6, June 2013 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2013, 41(6), 805–810.
RESEARCH PAPER
DNA Damage Detection by Electrochemiluminescence Sensor of CdS Quantum Dots LU Li-Ping*, XU Lai-Hui, KANG Tian-Fang, CHENG Shui-Yuan College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China
Abstract:
Electrochemiluminescence assay integrates the advantages of electrochemical potential controllability and high
sensitivity of luminescence analysis. The electrochemiluminescence sensor was fabricated to detect the DNA damage. CdS quantum dots were deposited on the surface of glass carbon electrode in 0.1 M CdCl2 and 0.02 M Na2S2O3 (0.1 M HCl, pH 2–3, 50 °C) by cyclic voltammetry, and then the amino-modified single strand DNA was immobilized on CdS modified electrode by the –CONH– bond using cysteine as linker agent. The electrochemiluminescence was used to study the interaction between perfluorooctane sulfonates and DNA, and H2O2 was used as the common reactant. The results demonstrated that the intensity of electrochemiluminescence signal increased with the increasing of perfluorooctane sulfonates concentration. Meanwhile, the electrochemical impedance spectroscopy indicated that the DNA charge transfer attenuated with the incubation of perfluorooctane sulfonates. These results reveal that perfluorooctane sulfonates can induce the distortion and change of DNA chain. Key Words: CdS quantum dots; Electrochemiluminescence; Perfluorooctane sulfonate; DNA damage
1
Introduction
Due to the unique physical and chemical properties, quantum dots (QDs) are a novel class of new luminescent materials, and they have been used in the fields of magnetics, optics, electricity, catalysis, chemical sensing and biomedicine etc. The synthetic methods of QDs mainly include precipitation method, hydrothermal synthesis method, microwave-assisted method, template method, electrodeposittion method and so on. The size of QDs can be controlled by regulating the depositional conditions in electrodeposition method. In recent reports, ITO glass[1–3] and Pt[4] are usually used as base materials to deposit QDs. However, there are few reports about the deposition of luminescent CdS QDs on the surface of glassy carbon electrode (GCE). On the other hand, the preparation methods of ELC sensors modified by CdS QDs mainly include the fabrication of carbon paste electrode[5] modified with CdS QDs, direct drop casting of CdS QDs on
the surface of electrode[6], the layer-by-layer self-assembly of CdS QDs on carbon electrode modified with carbon nanotube(CNT), and the assembly of CdS QDs on electrode through nuclide-avidin. The methods mentioned above are quite complicated because CdS QDs have to be pre-synthesized. In the present study, CdS QDs was modified onto electrode surface by a simple electrochemical deposition method. It is well known that DNA stores the genetic information, and therefore the maintenance of the integrity of DNA is of vital importance for cell function. Exogenous and endogenous factors can often lead to the loss of, or change of DNA molecules. A number of researches about DNA damage have been reported, and the reasons mainly include two aspects: the physical factors, such as the damages caused by ultraviolet radiation, and the chemical factors, such as alkylating agents. At present, the DNA damage is mainly detected by organisms bioculture and comet experimental detection[10–12].
Received 17 September 2012; accepted 26 December 2012 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 21005005), and the Beijing Nova Program of China (No. 2010B009); the Beijing Educational Committee of China (No. KM201010005014); the Deepening Plan of Talented Personnel in Beijing-Promising Key Projects, China (No.PHR20110818). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60659-3
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
Perfluorinated compounds (PFCs), especially perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been widely used in textiles, leathers, lubricants, electronic products and foam fire extinguishers, leading to the pollution of the global ecosystem and thus being a widespread concern in the global pollutants[13,14]. The high-energy carbon fluorine bonds in PFCs are strong enough to resist hydrolysis, light degradation, microbial metabolism and degradation, which make PFCs pollution have a high degree of persistence and bio-accumulation. The previous studies showed that PFCs could enter into organisms through respiratory tract, drinking water and food, and they are finally found in organisms of human beings, such as blood, liver, kidney and brain. At present, the poisonous mechanism of PFOS is still unclear and the research on molecure level is still scarce [20]. In this paper, the ECL was used to investigate the damage of DNA by PFOS.
2 2.1
Experimental
min. The cleaned GCE was immersed in a 0.2 M H2SO4 solution, and cyclic voltammetry (CV) scan was carried our between 1.3 V and 1.5 V. Then the GCE was washed by water and dried under purified nitrogen. The pretreated GCE was immersed in a 0.1 M CdCl2-0.02 M Na2S2O3 solution (0.1 M HCl, pH 2–3 at 50 °C), and then the CdS QDs were deposited on the surface of GCE by CV. The deposition conditions were as follows: the potential range was –0.2 V to –0.85 V (vs. SCE), the sweep speed was 0.05 V s–1 and the 30–50 cycles were performed. The whole deposition process was kept in a thermostatic water bath at 50 °C and thenthe CdS QDs/GCE was obtained. CdS QDs/GCE was immersed in L-cysteine solution at 4 °C for 6 h, and then the CdS QDs/GCE modified by L-cysteine was put into 1.0 μM NH2-ssDNA solution for 24 h, and ssDNA/L-CdS/GCE was obtained. DNA hybridization was realized by immersing the ssDNA/L-CdS/ GCE into the DNA solution for 30 min, and the dsDNA/ L-CdS/GCE was fabricated. dsDNA/L-CdS/GCE was immersed in the PFOS solution for 30 min at 37 °C.
Instruments and reagents 2.3
The apparatus were used as follows: CHI660A electrochemical workstation (Shanghai Chen Hua Instrument Corporation); photomultiplier (H9306-03, Hamamatsu), resistance value is 1 kΩ; three-electrode system: modified GCE (GCE, d = 3 mm）as working electrode, saturated calomel electrode(SCE) as reference electrode, platinum wire as counter electrode; KQ218 supersonic cleaner (Kunming Ultrasound Instrument Corporation); Atomic force microscope (AFM, SPA 300HV). CdCl2·5H2O was purchased from Beijing Chemical Reagent Corporation, Na2S2O3 from Beijing Chemical Reagent Corporation, Perfluorooctane sulfonate (PFOS, > 98.0%) from TCI Corporation, Tris-HCl from G&K, H2O2 from Beijing Chemical Reagent Corporation. All other chemicals were analytical reagent; all the solutions were prepared using double distilled- deionized water. DNA contains twenty oligonucleotide bases which were modified by 5'-NH2 and the target DNA was completely complementary pairing to the above probe, and the DNA was synthetized and purified by Shanghai Bioengineering Corporation. DNA sequences(5'to3') are as follows： DNA probe (ss-DNA): 5'-NH2-(CH2)6-GAC TCA CTA TAG GGA GGC GG-3'; Target DNA (T-DNA): 5'CCGCCTCCCTATAGTGAGTC-3'; DNA stock solution was prepared by TE (10 mM Tris-HCl + 1 mM EDTA pH 8.0). 2.2
Preparation of modified electrode
GCE was polished with 0.05 µm alumina/water slurry on a polishing pad to a mirror-like surface, followed by successive ultrasonic cleaning in ethanol and doubly distilled water for 2
Experimental methods
The modified electrode was immersed in 0.1 M phosphate buffer solution (PBS) + 38 mM H2O2 + 0.1 M KNO3 (or 0.1 M Tris-HCl (pH 8.0–8.8) + 0.1 M KNO3 + 38 mM H2O2) for cyclic voltammetry scanning with platinum filament as counter electrode and Ag/AgCl as reference electrode, and then PMT was cast under the electrode to collect optical signal. The electric resistance of PMT was set as 1 kΩ, and the range of electric potential was –1.7–0 V (vs. Ag/AgCl), and the potential scan rate was 50 mV s–1. The electrochemical impedance spectroscopy experiment: 0.1 M KCl, 10 mM K3Fe(CN)6-K4Fe(CN)6 (1:1, V/V) solution, frequency range: 1.0–105 Hz, the electric potential: 0.186 V.
3 3.1
Results and discussion AFM characterization of the DNA/L-CdS/GCE electrode interface
The morphologies of CdS QDs and the DNA/L-CdS modified on the surface of GCE were characterized by atomic force microscope (AFM). Figure 1 (left panel) shows the AFM image of CdS QDs modified electrode surface. It can be seen that the CdS nanopaticles is well deposited on the surface of electrode with the uniform diameter of 30 nm. When the DNA is assembled on the electrode surface (Fig.1. right panel), the surface morphology exhibits obvious change. Similar to the usual shape on the electrode surface, spherical DNA clusters apparently and intensely disperse on the electrode surface. The AFM measurements suggest that DNA has been successfully modified on the surface of CdS nanoparticles.
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
Fig.1 AFM images of CdS/GCE (left panel) and DNA/L-CdS/GCE (right panel)
3.2
Electrochemistry and ECL behavior of CdS QDs
To study the electrochemical behaviors of CdS and its ECL mechanism, the ECL signals of the CdS/GCE were monitored with or without the presence of hydrogen peroxide. Figure 2 shows the cyclic voltammograms of the modified electrode in different solution. By comparing curves a and b, it can be seen that an irreversible reduction peak of CdS QDs/GCE is formed at –1.1 V. However, the peak current in cureve a (with H2O2 solution) is significantly greater than that in curve b (without H2O2 solution). Meanwhile, the reduction current peak was not observed for the bare GCE electrode in the same electrolyte. Therefore, the CV measurements suggest that the reduction peak is probably concerned with the reduction of CdS NCs. The ECL curves of the modified electrode in different electrolyte were also compared. As seen in Fig.2B, the ECL signal can be clearly observed in the electrolyte with the presence of H2O2 at –1.1 V. However, the ECL signal was very weak in the solution without H2O2. The result indicated that compared with dissolved oxygen, the ECL intensity was significantly enhanced with H2O2 as a co-reactant. To further understand the ECL mechanism of CdS, the ECL of the GCE modified without sulfur source was also detected
in 0.1 M Tris-HCl (pH 8.0) + 0.1 M KNO3 + 38 mM H2O2 and no ECL signal was observed. Based on the above results, the luminescence mechanism can be inferred as follows: CdS + ne − → nCdS •− (1) •− 2CdS + H 2 O 2 → 2OH − + 2CdS∗ (2) CdS ∗ → CdS + hν
3.3
3.3.1
(3)
Optimization of the CdS/GCE ECL sensor detection system Effect of buffer solution
Figure 3 shows the ECL of the CdS/GCE in different buffer solutions. It can be seen that the ECL signal of CdS QDs/GCE is 1.55 V in the phosphate buffer solution (pH 9.0), and the ECL signal is approximately 0.11 V in Tris-HCl buffer solution (pH 8.0), which is 1.44 V lower than the former. From the comparison, 0.1 M PB (pH 9.0) + 0.1 M KNO3 was selected as the buffer solution in the present study. 3.3.2
Effect of CdS deposition cycles
The coverage and particle size of CdS on electrode surface
Fig.2 (A) Cyclic voltammograms of CdS/GCE in solution A (a) and solution B (b); and cyclic voltammogram of GCE in solution A (c). (A) 0.1 M Tris-HCl (pH 8.0) + 0.1 mol/L KNO3 + 38 mM H2O2; (B) 0.1 M Tris-HCl (pH 8.0) + 0.1 M KNO3. (B). ECL-potential curves of CdS/GCE in 38 mM H2O2 (a) , 0 M H2O2 (b) and Cd/GCE in 38 mM H2O2 (c) in 0.1 M Tris-HCl (pH 8.0) containing 0.1 M KNO3
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
electrode surface When damaged by PFOS the impedance of DNA decreased slightly, which is between those of the singleand double-stranded DNA. The reason is that the interactions between PFOS and DNA can lead to the distortion or breaking of DNA chain, which would decrease the coverage of DNA on the surface of CdS and increase the access probability of probe molecule to the surface of electrode, thus leading to the decreased impedance. 3.5
Fig.3
ECL-potential curves of CdS/GCE in 0.1 M PB (pH 9.0) (a) and 0.1 M Tris-HCl (pH 8.0) (b) containing 0.1 M KNO3 and 38 mM H2O2
were largely controlled by the electrodeposition cycles of CV, which strongly influenced the ECL of CdS QDs. Figure 4 shows the ECL curves of the CdS/GCE prepared with 30 (a) 40 (b) and 50 (c) CV cycles. It can be seen that the ECL value from the electrode treated with 40 deposition cycles was slightly larger than that from the electrode with 30 cycles, and the largest ECL value was obtained from the electrode with 50 cycles, which was 2.8 times larger than the former one. However, the thick modified layer could easily drop off from the electrode surface if more deposition cycles were used. On the other hand, the electrode treated with 50 cycles exhibited positive-shifted luminous potential. Based on the results, 50-cycle deposition was considered to be the best condition. 3.4
ECL of DNA/L-CdS/GCE
To investigate the DNA damage due to PFOS, the ECL signals of different modified electrodes were first analyzed in 0.1 M Tris-HCl (pH 8.0) + 0.1 M KNO3 + 38 mM H2O2. As shown in Fig.6, the CdS modified electrode produced the strongest ECL signal. The signal value of the modified electrode decreased with the single DNA and double DNA assembled on the electrode surface, and the ECL signal value was between those of the single-stranded and double-stranded DNA when the electrode was immersed in PFOS solution. Such result is consistent with the electrochemical impedance spectrum of the electrode surface, where the greater the
Electrochemical impedance characteristic of DNA/L-CdS/GCE
Impedance is usually used to characterize the charge transfer performance of modified electrode surface. An impedance spectrum usually consists of two parts, i.e. the process of high frequency controlled by dynamics, and the process of low frequency controlled by diffusion. Figure 5 shows the EIS curves of the different modified electrodes in 0.1 M KCl + 10 mM K3[Fe(CN)6]-K4[Fe(CN)6] (1:1, V/V). It can be seen that the impedance spectra exhibit a small semi-circle at low frequency, and the diameter of the semi-circle was about 200 Ω when the CdS NCs is modified, indicating the promoted charge transfer of probe molecule on the electrode surface . The impedance increased slightly to about 280 Ω after the single-DNA was modified and the impedance increased significantly after hybridized with complementary strand. The reason for the above results is that the double-stranded DNA contains more phosphate group than the single ones, which can hinder the access of the negative charge [Fe(CN)6]3–/4– to the surface of electrode. At the same time, the increased coverage of DNAcould significantly increase the access barriers of the probe molecule to the
Fig.4 ECL-potential curves of CdS/GCE for 30 (a), 40 (b) and 50 (c) sweep segments in 0.1 M PB (pH 9.0) containing 0.1 M KNO3 and 38 mM H2O2
a
Fig.5
b
d
c
Impedance spectra of CdS QDs/GCE (a), ssDNA/L-CdS/ GCE (b), dsDNA/L-CdS/GCE (c), PFOS-dsDNA/L-CdS/ GCE (d) in 10 mM K3Fe(CN)6-K4Fe(CN)6 (1:1, V/V) in 0.1 M KCl. CPFOS = 10 μM, 30 min, 37 °C
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
intensity of ECL increased with the PFOS concentration increasing. The reason may be that the higher PFOS concentration can lead to the increase of distortion or break of the surface DNA and the increase of naked surface area of CdS nanoparticles, resulting in the enhanced luminescent signals. It is also found that the PFOS concentration could be as low as 10–7 M.
References [1]
She X P, Di J W. Journal of Changshu Institute of Technology, 2010, 24(2): 71–75
Fig.6
ECL-potential curves of CdS/GCE (a), ssDNA/L-CdS/GCE (b), dsDNA/L-CdS/GCE (c), 10 µM PFOS-dsDNA/L-CdS/ GCE (d) in 0.1 M Tris-HCl (pH 9.0) containing 0.1 M KNO3 and 38 mM H2O2. 30 min, 37 °C
impedance is, the smaller the ECL value is. The increase of the electrode impedance can lead to the decrease of electron transfer, and therefore the ECL of CdS on the electrode surface is largely hampered. ECL values of different modified electrodes decreased in the order of CdS/GCE (110) > ssDNA/L-CdS/GCE (70) > dsDNA/L-CdS/GCE (25) (value unit: mV). After damaged by PFOS, the luminous intensity of the DNA and CdS modified on the electrode surface is between those of the single-stranded and double-stranded DNA, which reveals that the interaction of PFOS and DNA can lead to the distortion or breaking of DNA chains[24]. 3.6
Effect of PFOS concentration
Figure 7 shows the ECL spectra of the modified electrodes in 0.1 M Tris-HCl + 0.1 M KNO3 (pH 8.0) + 38 mM H2O2 after immersed in PFOS solution with different concentrations. It can be seen that the concentration of PFOS exhibits effect on the luminescence properties of the modified electrode. The
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Fig.7
ECL-potential curves of ssDNA/L-CdS/GCE (a), dsDNA/ L-CdS/GCE (b), 5 µM PFOS-dsDNA/L-CdS/GCE (c), 1 µM PFOS-dsDNA/L-CdS/GCE (d), 0.1 µM PFOS-dsDNA/ L-CdS/GCE (e) in 0.1 M PB (pH 9.0) containing 0.1 M KNO3 and 38 mDM H2O2. 30 min, 37 °C
[19] Tao L, Ma J, Kunisue T , Libelo E L, Tanabe S, Kannan K. Environmental
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8597–8602 [20] Fuentes S, Colomina M T, Vieens P, Domingo J L. Toxicology Letters, 2007: 171(3): 162–170
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
[21] Lu L P, Wang F, Kang T F, Cheng S Y, Wang Y T. Chinese J. Anal. Chem., 2011, 39: 392–396 [22] Brett A M O, Paquim A C. Bioelectrochemistry, 2005, 66: 117–124
[23] Shi C, Xu J J, Chen H Y. Journal of Electroanalytical Chemistry, 2007, 610(2): 186–192 [24] Lu L P, Xu L H, Kang T F, Cheng S Y. Biosensors and Bioelectronice, 2012, 35(1): 180–185
Cite this article as: Chin J Anal Chem, 2013, 41(6), 805–810.
RESEARCH PAPER
DNA Damage Detection by Electrochemiluminescence Sensor of CdS Quantum Dots LU Li-Ping*, XU Lai-Hui, KANG Tian-Fang, CHENG Shui-Yuan College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China
Abstract:
Electrochemiluminescence assay integrates the advantages of electrochemical potential controllability and high
sensitivity of luminescence analysis. The electrochemiluminescence sensor was fabricated to detect the DNA damage. CdS quantum dots were deposited on the surface of glass carbon electrode in 0.1 M CdCl2 and 0.02 M Na2S2O3 (0.1 M HCl, pH 2–3, 50 °C) by cyclic voltammetry, and then the amino-modified single strand DNA was immobilized on CdS modified electrode by the –CONH– bond using cysteine as linker agent. The electrochemiluminescence was used to study the interaction between perfluorooctane sulfonates and DNA, and H2O2 was used as the common reactant. The results demonstrated that the intensity of electrochemiluminescence signal increased with the increasing of perfluorooctane sulfonates concentration. Meanwhile, the electrochemical impedance spectroscopy indicated that the DNA charge transfer attenuated with the incubation of perfluorooctane sulfonates. These results reveal that perfluorooctane sulfonates can induce the distortion and change of DNA chain. Key Words: CdS quantum dots; Electrochemiluminescence; Perfluorooctane sulfonate; DNA damage
1
Introduction
Due to the unique physical and chemical properties, quantum dots (QDs) are a novel class of new luminescent materials, and they have been used in the fields of magnetics, optics, electricity, catalysis, chemical sensing and biomedicine etc. The synthetic methods of QDs mainly include precipitation method, hydrothermal synthesis method, microwave-assisted method, template method, electrodeposittion method and so on. The size of QDs can be controlled by regulating the depositional conditions in electrodeposition method. In recent reports, ITO glass[1–3] and Pt[4] are usually used as base materials to deposit QDs. However, there are few reports about the deposition of luminescent CdS QDs on the surface of glassy carbon electrode (GCE). On the other hand, the preparation methods of ELC sensors modified by CdS QDs mainly include the fabrication of carbon paste electrode[5] modified with CdS QDs, direct drop casting of CdS QDs on
the surface of electrode[6], the layer-by-layer self-assembly of CdS QDs on carbon electrode modified with carbon nanotube(CNT), and the assembly of CdS QDs on electrode through nuclide-avidin. The methods mentioned above are quite complicated because CdS QDs have to be pre-synthesized. In the present study, CdS QDs was modified onto electrode surface by a simple electrochemical deposition method. It is well known that DNA stores the genetic information, and therefore the maintenance of the integrity of DNA is of vital importance for cell function. Exogenous and endogenous factors can often lead to the loss of, or change of DNA molecules. A number of researches about DNA damage have been reported, and the reasons mainly include two aspects: the physical factors, such as the damages caused by ultraviolet radiation, and the chemical factors, such as alkylating agents. At present, the DNA damage is mainly detected by organisms bioculture and comet experimental detection[10–12].
Received 17 September 2012; accepted 26 December 2012 * Corresponding author. Email: [email protected] This work was supported by the National Natural Science Foundation of China (No. 21005005), and the Beijing Nova Program of China (No. 2010B009); the Beijing Educational Committee of China (No. KM201010005014); the Deepening Plan of Talented Personnel in Beijing-Promising Key Projects, China (No.PHR20110818). Copyright © 2013, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(13)60659-3
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
Perfluorinated compounds (PFCs), especially perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA), have been widely used in textiles, leathers, lubricants, electronic products and foam fire extinguishers, leading to the pollution of the global ecosystem and thus being a widespread concern in the global pollutants[13,14]. The high-energy carbon fluorine bonds in PFCs are strong enough to resist hydrolysis, light degradation, microbial metabolism and degradation, which make PFCs pollution have a high degree of persistence and bio-accumulation. The previous studies showed that PFCs could enter into organisms through respiratory tract, drinking water and food, and they are finally found in organisms of human beings, such as blood, liver, kidney and brain. At present, the poisonous mechanism of PFOS is still unclear and the research on molecure level is still scarce [20]. In this paper, the ECL was used to investigate the damage of DNA by PFOS.
2 2.1
Experimental
min. The cleaned GCE was immersed in a 0.2 M H2SO4 solution, and cyclic voltammetry (CV) scan was carried our between 1.3 V and 1.5 V. Then the GCE was washed by water and dried under purified nitrogen. The pretreated GCE was immersed in a 0.1 M CdCl2-0.02 M Na2S2O3 solution (0.1 M HCl, pH 2–3 at 50 °C), and then the CdS QDs were deposited on the surface of GCE by CV. The deposition conditions were as follows: the potential range was –0.2 V to –0.85 V (vs. SCE), the sweep speed was 0.05 V s–1 and the 30–50 cycles were performed. The whole deposition process was kept in a thermostatic water bath at 50 °C and thenthe CdS QDs/GCE was obtained. CdS QDs/GCE was immersed in L-cysteine solution at 4 °C for 6 h, and then the CdS QDs/GCE modified by L-cysteine was put into 1.0 μM NH2-ssDNA solution for 24 h, and ssDNA/L-CdS/GCE was obtained. DNA hybridization was realized by immersing the ssDNA/L-CdS/ GCE into the DNA solution for 30 min, and the dsDNA/ L-CdS/GCE was fabricated. dsDNA/L-CdS/GCE was immersed in the PFOS solution for 30 min at 37 °C.
Instruments and reagents 2.3
The apparatus were used as follows: CHI660A electrochemical workstation (Shanghai Chen Hua Instrument Corporation); photomultiplier (H9306-03, Hamamatsu), resistance value is 1 kΩ; three-electrode system: modified GCE (GCE, d = 3 mm）as working electrode, saturated calomel electrode(SCE) as reference electrode, platinum wire as counter electrode; KQ218 supersonic cleaner (Kunming Ultrasound Instrument Corporation); Atomic force microscope (AFM, SPA 300HV). CdCl2·5H2O was purchased from Beijing Chemical Reagent Corporation, Na2S2O3 from Beijing Chemical Reagent Corporation, Perfluorooctane sulfonate (PFOS, > 98.0%) from TCI Corporation, Tris-HCl from G&K, H2O2 from Beijing Chemical Reagent Corporation. All other chemicals were analytical reagent; all the solutions were prepared using double distilled- deionized water. DNA contains twenty oligonucleotide bases which were modified by 5'-NH2 and the target DNA was completely complementary pairing to the above probe, and the DNA was synthetized and purified by Shanghai Bioengineering Corporation. DNA sequences(5'to3') are as follows： DNA probe (ss-DNA): 5'-NH2-(CH2)6-GAC TCA CTA TAG GGA GGC GG-3'; Target DNA (T-DNA): 5'CCGCCTCCCTATAGTGAGTC-3'; DNA stock solution was prepared by TE (10 mM Tris-HCl + 1 mM EDTA pH 8.0). 2.2
Preparation of modified electrode
GCE was polished with 0.05 µm alumina/water slurry on a polishing pad to a mirror-like surface, followed by successive ultrasonic cleaning in ethanol and doubly distilled water for 2
Experimental methods
The modified electrode was immersed in 0.1 M phosphate buffer solution (PBS) + 38 mM H2O2 + 0.1 M KNO3 (or 0.1 M Tris-HCl (pH 8.0–8.8) + 0.1 M KNO3 + 38 mM H2O2) for cyclic voltammetry scanning with platinum filament as counter electrode and Ag/AgCl as reference electrode, and then PMT was cast under the electrode to collect optical signal. The electric resistance of PMT was set as 1 kΩ, and the range of electric potential was –1.7–0 V (vs. Ag/AgCl), and the potential scan rate was 50 mV s–1. The electrochemical impedance spectroscopy experiment: 0.1 M KCl, 10 mM K3Fe(CN)6-K4Fe(CN)6 (1:1, V/V) solution, frequency range: 1.0–105 Hz, the electric potential: 0.186 V.
3 3.1
Results and discussion AFM characterization of the DNA/L-CdS/GCE electrode interface
The morphologies of CdS QDs and the DNA/L-CdS modified on the surface of GCE were characterized by atomic force microscope (AFM). Figure 1 (left panel) shows the AFM image of CdS QDs modified electrode surface. It can be seen that the CdS nanopaticles is well deposited on the surface of electrode with the uniform diameter of 30 nm. When the DNA is assembled on the electrode surface (Fig.1. right panel), the surface morphology exhibits obvious change. Similar to the usual shape on the electrode surface, spherical DNA clusters apparently and intensely disperse on the electrode surface. The AFM measurements suggest that DNA has been successfully modified on the surface of CdS nanoparticles.
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
Fig.1 AFM images of CdS/GCE (left panel) and DNA/L-CdS/GCE (right panel)
3.2
Electrochemistry and ECL behavior of CdS QDs
To study the electrochemical behaviors of CdS and its ECL mechanism, the ECL signals of the CdS/GCE were monitored with or without the presence of hydrogen peroxide. Figure 2 shows the cyclic voltammograms of the modified electrode in different solution. By comparing curves a and b, it can be seen that an irreversible reduction peak of CdS QDs/GCE is formed at –1.1 V. However, the peak current in cureve a (with H2O2 solution) is significantly greater than that in curve b (without H2O2 solution). Meanwhile, the reduction current peak was not observed for the bare GCE electrode in the same electrolyte. Therefore, the CV measurements suggest that the reduction peak is probably concerned with the reduction of CdS NCs. The ECL curves of the modified electrode in different electrolyte were also compared. As seen in Fig.2B, the ECL signal can be clearly observed in the electrolyte with the presence of H2O2 at –1.1 V. However, the ECL signal was very weak in the solution without H2O2. The result indicated that compared with dissolved oxygen, the ECL intensity was significantly enhanced with H2O2 as a co-reactant. To further understand the ECL mechanism of CdS, the ECL of the GCE modified without sulfur source was also detected
in 0.1 M Tris-HCl (pH 8.0) + 0.1 M KNO3 + 38 mM H2O2 and no ECL signal was observed. Based on the above results, the luminescence mechanism can be inferred as follows: CdS + ne − → nCdS •− (1) •− 2CdS + H 2 O 2 → 2OH − + 2CdS∗ (2) CdS ∗ → CdS + hν
3.3
3.3.1
(3)
Optimization of the CdS/GCE ECL sensor detection system Effect of buffer solution
Figure 3 shows the ECL of the CdS/GCE in different buffer solutions. It can be seen that the ECL signal of CdS QDs/GCE is 1.55 V in the phosphate buffer solution (pH 9.0), and the ECL signal is approximately 0.11 V in Tris-HCl buffer solution (pH 8.0), which is 1.44 V lower than the former. From the comparison, 0.1 M PB (pH 9.0) + 0.1 M KNO3 was selected as the buffer solution in the present study. 3.3.2
Effect of CdS deposition cycles
The coverage and particle size of CdS on electrode surface
Fig.2 (A) Cyclic voltammograms of CdS/GCE in solution A (a) and solution B (b); and cyclic voltammogram of GCE in solution A (c). (A) 0.1 M Tris-HCl (pH 8.0) + 0.1 mol/L KNO3 + 38 mM H2O2; (B) 0.1 M Tris-HCl (pH 8.0) + 0.1 M KNO3. (B). ECL-potential curves of CdS/GCE in 38 mM H2O2 (a) , 0 M H2O2 (b) and Cd/GCE in 38 mM H2O2 (c) in 0.1 M Tris-HCl (pH 8.0) containing 0.1 M KNO3
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
electrode surface When damaged by PFOS the impedance of DNA decreased slightly, which is between those of the singleand double-stranded DNA. The reason is that the interactions between PFOS and DNA can lead to the distortion or breaking of DNA chain, which would decrease the coverage of DNA on the surface of CdS and increase the access probability of probe molecule to the surface of electrode, thus leading to the decreased impedance. 3.5
Fig.3
ECL-potential curves of CdS/GCE in 0.1 M PB (pH 9.0) (a) and 0.1 M Tris-HCl (pH 8.0) (b) containing 0.1 M KNO3 and 38 mM H2O2
were largely controlled by the electrodeposition cycles of CV, which strongly influenced the ECL of CdS QDs. Figure 4 shows the ECL curves of the CdS/GCE prepared with 30 (a) 40 (b) and 50 (c) CV cycles. It can be seen that the ECL value from the electrode treated with 40 deposition cycles was slightly larger than that from the electrode with 30 cycles, and the largest ECL value was obtained from the electrode with 50 cycles, which was 2.8 times larger than the former one. However, the thick modified layer could easily drop off from the electrode surface if more deposition cycles were used. On the other hand, the electrode treated with 50 cycles exhibited positive-shifted luminous potential. Based on the results, 50-cycle deposition was considered to be the best condition. 3.4
ECL of DNA/L-CdS/GCE
To investigate the DNA damage due to PFOS, the ECL signals of different modified electrodes were first analyzed in 0.1 M Tris-HCl (pH 8.0) + 0.1 M KNO3 + 38 mM H2O2. As shown in Fig.6, the CdS modified electrode produced the strongest ECL signal. The signal value of the modified electrode decreased with the single DNA and double DNA assembled on the electrode surface, and the ECL signal value was between those of the single-stranded and double-stranded DNA when the electrode was immersed in PFOS solution. Such result is consistent with the electrochemical impedance spectrum of the electrode surface, where the greater the
Electrochemical impedance characteristic of DNA/L-CdS/GCE
Impedance is usually used to characterize the charge transfer performance of modified electrode surface. An impedance spectrum usually consists of two parts, i.e. the process of high frequency controlled by dynamics, and the process of low frequency controlled by diffusion. Figure 5 shows the EIS curves of the different modified electrodes in 0.1 M KCl + 10 mM K3[Fe(CN)6]-K4[Fe(CN)6] (1:1, V/V). It can be seen that the impedance spectra exhibit a small semi-circle at low frequency, and the diameter of the semi-circle was about 200 Ω when the CdS NCs is modified, indicating the promoted charge transfer of probe molecule on the electrode surface . The impedance increased slightly to about 280 Ω after the single-DNA was modified and the impedance increased significantly after hybridized with complementary strand. The reason for the above results is that the double-stranded DNA contains more phosphate group than the single ones, which can hinder the access of the negative charge [Fe(CN)6]3–/4– to the surface of electrode. At the same time, the increased coverage of DNAcould significantly increase the access barriers of the probe molecule to the
Fig.4 ECL-potential curves of CdS/GCE for 30 (a), 40 (b) and 50 (c) sweep segments in 0.1 M PB (pH 9.0) containing 0.1 M KNO3 and 38 mM H2O2
a
Fig.5
b
d
c
Impedance spectra of CdS QDs/GCE (a), ssDNA/L-CdS/ GCE (b), dsDNA/L-CdS/GCE (c), PFOS-dsDNA/L-CdS/ GCE (d) in 10 mM K3Fe(CN)6-K4Fe(CN)6 (1:1, V/V) in 0.1 M KCl. CPFOS = 10 μM, 30 min, 37 °C
LU Li-Ping et al. / Chinese Journal of Analytical Chemistry, 2013, 41(6): 805–810
intensity of ECL increased with the PFOS concentration increasing. The reason may be that the higher PFOS concentration can lead to the increase of distortion or break of the surface DNA and the increase of naked surface area of CdS nanoparticles, resulting in the enhanced luminescent signals. It is also found that the PFOS concentration could be as low as 10–7 M.
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Fig.6
ECL-potential curves of CdS/GCE (a), ssDNA/L-CdS/GCE (b), dsDNA/L-CdS/GCE (c), 10 µM PFOS-dsDNA/L-CdS/ GCE (d) in 0.1 M Tris-HCl (pH 9.0) containing 0.1 M KNO3 and 38 mM H2O2. 30 min, 37 °C
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Fig.7
ECL-potential curves of ssDNA/L-CdS/GCE (a), dsDNA/ L-CdS/GCE (b), 5 µM PFOS-dsDNA/L-CdS/GCE (c), 1 µM PFOS-dsDNA/L-CdS/GCE (d), 0.1 µM PFOS-dsDNA/ L-CdS/GCE (e) in 0.1 M PB (pH 9.0) containing 0.1 M KNO3 and 38 mDM H2O2. 30 min, 37 °C
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