Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples

Chinese Chemical Letters 25 (2014) 29–34

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Chinese Chemical Letters journal homepage: www.elsevier.com/locate/cclet

Original article

Electrochemical DNA nano-biosensor for the detection of genotoxins in water samples Hong-Bo Xu a,b, Ran-Feng Ye b, Shang-Yue Yang b, Rui Li b, Xu Yang b,* a b

Wuhan No. 11 High School, Wuhan 430030, China Lab of Environmental Biomedicine, Central China Normal University, Wuhan 430079, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 June 2013 Received in revised form 18 August 2013 Accepted 16 September 2013 Available online 12 November 2013

In the present study, a disposable electrochemical DNA nano-biosensor is proposed for the rapid detection of genotoxic compounds and bio-analysis of water pollution. The DNA nano-biosensor is prepared by immobilizing DNA on Au nanoparticles and a self-assembled monolayer of cysteamine modified Au electrode. The assembly processes of cysteamine, Au nanoparticles and DNA were characterized by cyclic voltammetry (CV). The Au nanoparticles enhanced DNA immobilization resulting in an increased guanine signal. The interaction of the analyte with the immobilized DNA was measured through the variation of the electrochemical signal of guanine by square wave voltammetry (SWV). The biosensor was able to detect the known genotoxic compounds: 2-anthramine, acridine orange and 2naphthylamine with detection limits of 2, 3 and 50 nmol/L, respectively. The biosensor was also used to test actual water samples to evaluate the contamination level. Additionally, the comparison of results from the classical genotoxicity bioassay has confirmed the applicability of the method for real samples. ß 2013 Xu Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.

Keywords: Electrochemical DNA biosensor Au nanoparticles Genotoxic detection DNA damage

1. Introduction More and more pollutants exhibiting genotoxicity have invaded the environment in recent decades, which are threatening human health. Genotoxic contaminants from industrial effluents are often discharged into surface water, and then later used downstream for agricultural irrigation, or even as sources of drinking water. Many studies have shown the presence of several organic mutagens and carcinogens in drinking waters, and epidemiological studies have highlighted the correlation between genotoxicity of drinking water and increased risks of cancer [1–4]. Therefore, it is essential to establish simple and rapid screening techniques to determine the presence of genotoxins and evaluate the effects of water quality on human health. Currently, the pollutants can be determined by gas chromatography, high performance liquid chromatography or spectroscopy, which offer the possibility of identifying and quantifying specific compounds with high resolution and good precision. However, these chemical measurements cannot reflect the real effects of genotoxicity because genotoxicity is a biological response. Only bioassays are able to evaluate the combined action from potentially hazardous compounds as complex mixtures in environment. To date, several classic bioassays have been

* Corresponding author. E-mail address: [email protected] (X. Yang).

established for genotoxicity analysis, including the Salmonella typhimurium mutagenicity test with strains TA98 and/or TA100 (Ames test) [5,6], single-cell gel electrophoretic (SCGE or Comet) assay [7,8], micronucleus assay [9] and so on. The genotoxicity bioassays can determine the effects of hazardous compounds on nuclear DNA, providing direct and appropriate measurements of genotoxicity, but they are time-consuming, can be costly and analyses can require several days or weeks. Compared with these methods, the electrochemical DNA biosensor may be an ideal candidate for screening of these pollutants, as they require a small amount of sample and are based on biological interaction [10]. The most critical step in the preparation of DNA electrochemical biosensors is the immobilization of DNA strands on the surface of an electrode. The amount of immobilized DNA directly affects the accuracy, sensitivity and selectivity of the DNA electrochemical sensors [11]. In recent years, there has been great progress in the application of nanomaterials in biosensors to elevate sensitivity and enhance electrochemical performance [12–15]. Additionally, the high surface-to-volume ratio of Au nanoparticles can assemble larger volumes of DNA on the electrode surface [16,17]. The present study aimed at developing a more sensitive electrochemical DNA biosensor for the detection of genotoxins in water samples. A monolayer of cysteamine was self-assembled on the Au electrode surface first, and then Au nanoparticles were assembled on it. After the thiolated ssDNA were immobilized onto Au nanoparticles to form the ssDNA/Nano/Au, the dsDNA/Nano/Au biosensor was constructed by the hybridization of complementary

1001-8417/$ – see front matter ß 2013 Xu Yang. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved. http://dx.doi.org/10.1016/j.cclet.2013.10.011

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H.-B. Xu et al. / Chinese Chemical Letters 25 (2014) 29–34

DNA onto the thiolated ssDNA. Then, the degree of oxidative damage caused to DNA was monitored by the change of the peak current as a consequence of guanine oxidation. After comparison between the performances of the ssDNA/Nano/Au and the dsDNA/ Nano/Au on standard 2-anthramine, the dsDNA/Nano/Au was used as the DNA nano-biosensor to test actual water samples. 2. Experimental The synthetic DNA oligonucleiotides were purchased from Sangon Biological Engineering Technology & Co. Ltd. (Shanghai, China). The base sequences are as follows [18]: thiol-terminated DNA probe: 50 -SH–(CH2)6–CAG GCG GCC GCA CAC GCC TCC A-30 , complementary target: 50 -TGG AGG ACG TGT GCG GCC GCC TG-30 . Stock solutions of the oligonucleotides were dissolved in 20 mmol/L Tris(hydroxymethyl)aminomethane (tris)–HCl (pH 8.0) containing 100 mmol/L MgCl2, and kept refrigerated at 20 8C. De-ionized water was used throughout all experiments. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl43H2O) (99.9%), trisodium citrate, 2-anthramine, acridine orange and 2naphthylamine were obtained from Sigma–Aldrich Company (USA). All the reagents were analytical grade and were used as received. A JEM-2100 transmission electron microscope (TEM) (JEOL, Japan) operating at 200 kV was used to size the Au nanoparticles. Electrochemical experiments were performed on A CHI 660 C Electrochemical Workstation (Shanghai CH Instrument Company, China) with a three-electrode system using the modified Au electrode as the working electrode, a platinum wire as the counter electrode and a saturated calomel reference electrode (SCE) as the reference electrode. All the potentials were referred to the SCE reference electrode. Water samples (X1-3) were collected at three sites from a village in Henan Province, China. Each water sample (3 L) was firstly filtered under vacuum using a glass fiber filter with a 0.22 mm pore size. The filtered water samples were then acidified (pH 3.0) with 20% acetic acid and passed through pre-conditioned Oasis HLB cartridges (200 mg/6cc, 30 mm partial size, Waters Corporation, USA) at a rate of approximately 3 mL/min. Each sample was eluted with acetone (11 mL), and then evaporated to dryness under a gentle nitrogen stream at 37 8C. The residue was immediately dissolved in 1 mL dimethylsulfoxide (DMSO) as stock extract solution. Three dilutions were made from the stock extract solutions for genotoxicity analysis, in which each 1 mL was equivalent to 25, 50 or 100 mL of the original water source, respectively (the dosages were described as 25, 50 or 100).

Fig. 1. The TEM image of the Au nanoparticles.

the electrode was washed with ultra pure water and dried under a pure nitrogen stream. The procedures for assembling the Au nanoparticles onto the Au electrode surface were according to the literature [17] with only slight modification. Briefly, the cleaned electrode was initially immersed into 10 mmol/L cysteamine for 2 h under ambient conditions to allow the self-assembly of cysteamine, followed by immersion into the Au nanoparticle solution for 10 h. After this period, the electrode was washed with water to remove unbounded Au nanoparticles. The steps for DNA immobilization and DNA hybridization were according to the previous literature with a minor modification [18]. The electrode modified with Au nanoparticles was immersed in 15 mL of 2 mmol/L thiolated ssDNA at 4 8C for 6 h to obtain the ssDNA/Nano/Au. Then the ssDNA/nano/Au was rinsed with water to avoid the physical absorption. The dsDNA/Nano/Au was prepared by pipetting 15 mL of 2 mmol/L complementary DNA onto the ssDNA/Nano/Au, and kept for 2 h to perform DNA hybridization at room temperature. Subsequently, the obtained dsDNA/Nano/Au was washed with water and stored at 4 8C until use. The modified electrodes were characterized by electrochemical scanning in 2.5 mmol/L K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) solution containing 0.1 mol/L KCl. Cyclic voltammetry (CV) measurements were swept from 0 V to 0.6 V with a sweeping rate of 0.1 Vs. For comparison with and without Au nanoparticles modification, the cleaned Au electrode was immersed in 15 mL of 2 mmol/L thiolated ssDNA solution under the same condition to obtain the ssDNA/Au. The dsDNA/Au was also prepared by pipetting 15 mL of 2 mmol/L complementary DNA onto the ssDNA/Au.

2.1. Au nanoparticles preparation 2.3. Detection of genotoxic compounds with DNA-nano biosensor The Au nanoparticles were prepared by the citrate reduction of HAuCl4 according to the literature [19]. Briefly, 10 mL of 38.8 mmol/L trisodium citrate was added to 100 mL of boiling 1.0 mmol/L HAuCl4 solution and stirred for 15 min at the boiling point. The solution color turned to wine red, indicating the formation of Au nanoparticles. Then, the solution was allowed to cool to room temperature and stored in a dark bottle at 4 8C. The prepared Au nanoparticles have an average diameter of approximately 15 nm measured by TEM (Fig. 1). 2.2. Preparation and characterization of the modified electrode The Au electrode surface was polished with 1 mm, 0.3 mm and 0.05 mm a-Al2O3 powder, and washed with de-ionized water and 95% ethanol. Before surface modification, the bare Au electrode was scanned in 0.5 mol/L H2SO4 between 0.3 V and 1.5 V until a reproducible cyclic voltammogram was achieved. Following that,

The peak current of guanine was used as the transduction signal for recognizing DNA interacting agents, and the current signal was measured by square wave voltammetry (SWV) in 0.5 mol/L acetate buffer solution (pH 4.7, containing 10 mmol/L of sodium chloride) at room temperature. For SWV measurements, a potential range between +0.5 V and +1.2 V, frequency of 200 Hz, step potential of 15 mV and amplitude of 40 mV were applied. The volume of the electrochemical cell used in the study was 20 mL. As the result of interaction of DNA with a genotoxic compound, a decrease of guanine peak current was observed. The decrease percentage of guanine peak current (Dpc) was used to evaluate the potentially toxic analytes: Dpc = [1  (Is/Ib)]  100%, where Ib is the peak current of DNA-nano-biosensor scanned in acetate buffer before treatment with the analyte, and Is is the peak current of the DNA-nano biosensor after incubated with the analyte solution for 20 min.

H.-B. Xu et al. / Chinese Chemical Letters 25 (2014) 29–34

2.4. Ames test and Comet assay

26

In order to study the applicability of the biosensor for actual environmental samples, genotoxicity evaluations of the water samples were also performed by studying with the Ames test and the Comet assay. The Ames test was performed on filtered extracts in triplicate at increasing concentrated dosage of water samples per plate, using the S. typhimurium TA98 with S9 mix according to the direct plateincorporation method [6]. For 48 h incubation of agar plates at 37 8C, bacterial colonies were counted. The resulting data were the average of triplicate plates and were expressed as mutagenicity ratio (M.R.), dividing the revertants/plate by the spontaneous mutation rate, M.R. = (revertants/plate for sample)/(revertants/ plate for blank control). The Comet assay was essentially performed according to the literature with the human HepG2 cell line [7]. Briefly, frosted microscope slides, on which cells were embedded in an agarose sandwich, were put into cell lysis solution. After DNA was allowed to unwind in an electrophoresis buffer, slides were placed into a horizontal electrophoresis tank and exposed to 25 V and 300 mA for 30 min. Then, the DNA on the microscope slides was stained with ethidium bromide (EB) and 50 randomly selected per coded slides were examined with a fluorescent microscope (BX41, Olympus, Japan). Among the parameters available for the analysis of the extent of DNA damage, Olive Tail Moment (OTM) was chosen as the most relevant measure of genotoxicity [20]. OTM, defined as tail length and the percentage of DNA in comet tails and heads, were calculated by CASP software [21].

18

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2.5. Statistical analysis A one-way analysis of variance (ANOVA) was used to compare between the different sample groups. Dunnett’s test was used to determine the significance of differences between the sample groups and the control (blank) group. Statistical analysis was performed with Statistical Product and Service solutions (SPSS 11.0). A significance level of p < 0.05 was applied in all statistical tests. 3. Results and discussion

I (μA)

a b c

10

d

e

2 -6 -14 -22 0.6

0.5

0.4

0.3

0.2

0.1

0

-0.1

-0.2

E (V) Fig. 2. CVs of the bare Au electrode (a), Au nanoparticles and cysteamine monolayer modified Au electrode (b), cysteamine monolayer modified Au electrode (c), ssDNANano/Au (d), dsDNA-Nano/Au (e) in 2.5 mmol/L [Fe(CN)6]3/4 aqueous solution containing 0.1 mmol/L KCl at a scan rate of 100 mV/s.

repellence of [Fe(CN)6]3/4 by the negatively charged phosphate backbone, implying that ssDNA has been successfully immobilized on the surface of the modified electrode. Here, we selected thiolterminated ssDNA to modify the electrode because of the strong affinity of thiol-metal (SH-Au) linkages [24]. The redox peaks of [Fe(CN)6]3/4 were further decreased when the ssDNA/nano/Au was subsequently hybridized with complementary DNA (Fig. 2e), indicating that double-stranded DNA have been formed on the electrode surface to obtain the dsDNA/nano/Au electrode. Additionally in the CVs, there is an increasing peak-to-peak separation from curve (a) to curve (e), which indicates that the kinetics of the electron transfer slows down with the step wise electrode modification. 3.2. Electrochemical behavior of DNA on the modified electrodes In order to study the electrochemical behavior of DNA on the modified electrodes, the changes in DNA redox properties were monitored by SWV method (Fig. 3). We employed SWV because it has been demonstrated that this electrochemical technique is very sensitive for nucleic acids detection and combines the effective

3.1. Cyclic voltammetry of [Fe(CN)6]3/4 at modified electrode 5.0 e 4.0

d

3.0 I (μA)

The electron transfer of ferricyanide through the modified electrode could be used as a valuable tool to monitor the entire electrode fabrication process. Fig. 2 displays the cyclic voltammetric signals of [Fe(CN)6]3/4 on the bare Au electrode and differently modified electrodes. A pair of obvious redox peaks, which could be ascribed to the redox behaviors of [Fe(CN)6]3/4, were observed on the bare Au electrode (Fig. 2a). After the self-assembly of cysteamine monolayer was formed on the Au electrode surface, there was a small decrease of peak currents (Fig. 2c), which was invoked by the diffusion inhibition of [Fe(CN)6]3/ to the electrode surface. Then, Au nanoparticles were assembled on the electrode surface through its electrostatic interaction with cysteamine, so a slight increase in the voltammetric response of [Fe(CN)6]3/4 was observed (Fig. 2b). This enhanced electrochemical performance was attributed to the presence of Au nanoparticles modification, which was convinced to enable an increase of the effective electrode surface area and enhance the rate of electron transfer [22,23]. Compared to the Au electrode modified with Au nanoparticles and cysteamine monolayer (Fig. 2b), when the electrode surface was further covered by thiol-terminated ssDNA to form the ssDNA/ nano/Au (Fig. 2d), the redox peaks were decreased due to the

2.0

c b

1.0

a 0.0 0.5

0.7

0.9

1.1

E (V) Fig. 3. SWV for the bare Au (a), dsDNA/Au (b), ssDNA/Au (c), dsDNA/Nano/Au (d) and ssDNA/Nano/Au (e) in acetate buffer (pH 4.7) with 10 mmol/L NaCl. Potential range from +0.5 V to +1.2 V, frequency = 200 Hz, step = 15 mV, amplitude = 40 mV. The concentration of thiol-terminated ssDNA immobilized onto electrodes surface was 2 mmol/L.

H.-B. Xu et al. / Chinese Chemical Letters 25 (2014) 29–34

charging-current compensation with a rapid scanning capability [25]. As shown in Fig. 3, the bare Au electrode shows a flat curve (Fig. 3a). When the electrodes were modified with DNA, sharp oxidation peaks of guanine were observed at about +0.9 V (Fig. 3b– e), which were in agreement with +0.9 V for guanine reported by others [26]. Compared with the signal obtained with the ssDNA/Au (Fig. 2c), the ssDNA/Nano/Au (Fig. 3e) produced an obvious signal increase. Also, the peak current obtained with the dsDNA/Nano/Au (Fig. 3d) was higher than with the dsDNA/Au (Fig. 3b). This may be because Au nanoparticles enhance the electron transfer signal of the immobilized DNA, and the high surface-to-volume ratio characteristic of Au nanoparticles can increase the amount thiol-terminated ssDNA immobilization [27]. Meanwhile, the guanine signal obtained with the ssDNA/Nano/ Au (Fig. 3e) was higher than that with the dsDNA/Nano/Au (Fig. 3d) and the signal obtained with the ssDNA/Au (Fig. 3c) was higher than that with the dsDNA/Au (Fig. 3b), which could be attributed to the increased availability for oxidation of guanine bases in singlestranded DNA than in double-stranded DNA [28]. 3.3. Comparison of analytical performances with the dsDNA/Nano/Au and the ssDNA/nano/Au Since the electrodes modified with single-strand DNA, or double-strand DNA, can produce different guanine peak currents, they may have different response to genotoxic compounds. Therefore, 2-anthramine, commonly used as positive DNA damage standard in the research of DNA biosensors [28,29], was used to evaluate the performance of the ssDNA/Nano/Au and the dsDNA/ Nano/Au biosensors. From Fig. 4, the guanine peak current obtained with the ssDNA/ Nano/Au (Fig. 4a) was higher than with the dsDNA/Nano/Au (Fig. 4b). After the two biosensors were incubated with 2anthramine for 20 min, the resulting anodic peaks of the ssDNA/ Nano/Au (Fig. 4c) and the dsDNA/Nano/Au (Fig. 4d) were decreased. The decrease percentage of peak current (Dpc) could be proportional to the different concentration of 2-anthramine. As shown in Fig. 5, the dsDNA/Nano/Au biosensor exhibited a linear response to 2-anthramine concentration ranging from 10 nmol/L to 1.7  103 nmol/L with the slope 40.514 (r = 0.995). 5.0 a b

3.0

c

I (μA)

4.0

d

100

80

60

I p%

32

40

20

0 0

0.5 1 2-Anthramine (μmol/L)

1.5

Fig. 5. Calibration plot of the decrease percentage in the guanine oxidation peak current (Dpc) with the different concentration of 2-anthramine at the ssDNA/Nano/ Au (&) and the dsDNA/Nano/Au (&). The dotted lines are linear fits of the data.

The detection limit was 2 nmol/L at a signal-to-noise ratio of 3. The linear range of the ssDNA/Nano/Au for 2-anthramine concentration was 10 nmol/L to 1.1  103 nmol/L with the detection limit of 2 nmol/L and the slope for linear regression 56.524 (r = 0.991). In fact, when using the ssDNA/Nano/Au, as the concentration of 2anthramine increased above 1.2  103 nmol/L, Dpc did not proportionally increase and gradually approached saturation. From these results, both of the two biosensors have higher sensitivity for 2-anthramine than the other DNA biosensors fabricated by directly adsorbing DNA on the surface of electrodes [28,29]. Although a higher current intensity was obtained with the ssDNA/Nano/Au, the linear range observed for 2-anthramine with the dsDNA/Nano/Au was wider while both of the two biosensors had similar detection limits. Therefore, the dsDNA/Nano/Au was chosen as the model to do the following experiments. The repeatability of the electrochemical measurements of DNA guanine peak current was estimated as less than 8% of relative standard deviation (RSD). The dsDNA/Nano/Au biosensor was also used to study the response to other DNA damage compounds, such as acridine orange and 2-naphthylamine, which are known as genotoxic. Incubation with these chemicals resulted in a decrease in guanine peak current signal. A summary of analytical parameters were listed in Table 1, including the slope, the linear range, the intercept of the linear fitting and the limits of detections (LOD). In order to confirm the biosensor specificity to genotoxins, the biosensor was also treated with non-genotoxic compounds, including phenol and cyclohexane, which resulted in little response.

2.0

3.4. Application for detection of environmental water samples 1.0

0.0 0.5

0.7

0.9

1.1

E (V) Fig. 4. SWV curves of the ssDNA-Nano/Au and the dsDNA/Nano/Au obtained in acetate buffer (pH 4.7) with 10 mmol/L NaCl. Potential range from +0.5 V to +1.2 V, frequency = 200 Hz, step = 15 mV, amplitude = 40 mV: the ssDNA/Nano/Au biosensor signal (blank) (a); the dsDNA/Nano/Au biosensor signal (blank) (b); the dsDNA/Nano/Au biosensor after incubated with 500 nmol/L 2-anthramine (c); the ssDNA/Nano/Au biosensor after incubated with 500 nmol/L 2-anthramine (d). Incubation time = 20 min.

In order to compare the performance of the biosensor on water samples with other genotoxicity methods, the Ames test and the Comet assay, which are the two major tools for genotoxicity analysis of environmental pollution, were used in this study. For the biosensor, if a sample had a Dpc > 15% (calculated by signal-to-noise ratio of 3), it was considered genotoxic. According to the EPA guidelines of the Ames test [30], a genotoxic/mutagenic potential of a test sample is confirmed if the mutant ratio (M.R.) is 2.0 or higher. In the Comet assay, if there was a statistically significant difference in OTM between the sample and the blank control group (OTM = 0.31  0.11), the water sample was identified as potentially genotoxic.

H.-B. Xu et al. / Chinese Chemical Letters 25 (2014) 29–34

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Table 1 Analytical features obtained for the standard curves of acridine orange and 2-naphthylamine by the dsDNA/nano/Au biosensor. Analytes Acridine orange 2-Naphthylamine

Linear range (nmol/L) 10–900 100–2500

Slope (mA/nmol/L) 3

74.105  10 1.594  103

Intercept (mA)

Correlation coefficient (n = 5)

LOD (nmol/L)

12.475 12.958

0.990 0.994

3 50

Table 2 Comparison of results from the dsDNA-nano biosensor and other genotoxicity methods for detection of water samples. Samples

Dosage

Ames test (M.R.) TA98

Comet assay (OTM, x¯  s)

X1

25 50 100

40.18  6.00 48.61  5.78 69.35  6.34

2.04  0.43 2.64  0.37 3.04  1.01

2.88  0.41* 3.09  0.59* 4.53  0.46*

X2

25 50 100

15.09  3.21 25.32  4.27 32.18  5.04

1.17  0.32 1.38  0.35 1.52  0.51

0.42  0.27 0.88  0.23* 1.13  0.27*

X3

25 50 100

11.52  4.80 16. 89  3.01 29.20  3.89

1.01  0.23 1.22  0.31 1.37  0.29

0.37  0.19 0.65  0.21* 1.08  0.21*

dsDNA/nano biosensor (Dpc, %)

Data are expressed as x¯  SEM (n = 3). * p < 0.05 compared with blank control (OTM = 0.31  0.11).

From Table 2, the three methods showed an increase in DNA damage with the higher concentrated level of water samples. The water sample, X1, was demonstrated with genotoxicity at all three concentrated levels by the biosensor and the other two genotoxicity bioassays. The water samples X2 and X3 were tested as having no genotoxic/mutagenic potential by the Ames test. However, the Comet assay demonstrated X2 and X3 water samples containing genotoxic compounds at 50 concentrated level. This may be attributed to the fact that the Comet assay is more sensitive than the Ames test [31]. Also, the dsDNA/Nano/Au biosensor showed similar results to the sensitive comet assay that X2 and X3 water samples were identified with genotoxicity at the 50 concentrated level. From the results, the biosensor could be used to distinguish different contamination level. 4. Conclusion In the present work, a new system has been developed for the rapid bio-anaylsis of genotoxic compounds based on the electrochemical DNA-nano-biosensor. The modification of Au nanoparticles on the electrode surface enhanced DNA immobilization and increased the electrochemical signal. Furthermore, the dsDNA/ Nano/Au biosensor can distinguish different contamination levels of water samples and show good correlations of results with the Comet assay. The analysis of a sample can be performed in minutes instead of days without dependence on cell or bacteria. Therefore, this kind of biosensor could be very useful as an easy and fast screening method for genotoxicity analysis of environment pollution. Acknowledgments This work was funded by the National Natural Science Foundation of China (Nos. 21103059, 51136002 and 51076079) and the China Key Technologies R&D Program (No. 2012BAJ02B03). References [1] M. Argos, T. Kalra, P.J. Rathouz, et al., Arsenic exposure from drinking water, and all-cause and chronic-disease mortalities in Bangladesh (HEALS): a prospective cohort study, Lancet 376 (2010) 252–258. [2] H.P. Yu, L.Y. Shi, W.H. Lu, et al., Expression of cyclooxygenase-2 (COX-2) in human esophageal cancer and in vitro inhibition by a specific COX-2 inhibitor, NS-398, J. Gastroenterol. Hepatol. 19 (2004) 638–642.

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