Electrochemical Sensor Based on Electrodeposited Graphene-Au

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Electrochemical Sensor Based on Electrodeposited Graphene-Au Modified Electrode and NanoAu Carrier Amplified Signal Strategy for Attomolar Mercury Detection Yi Zhang, Guangming Zeng, Lin Tang, Jun Chen, Yuan Zhu, Xiaoxiao He, and Yan He Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503472p • Publication Date (Web): 02 Dec 2014 Downloaded from http://pubs.acs.org on December 3, 2014

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Analytical Chemistry

Electrochemical Sensor Based on Electrodeposited Graphene-Au Modified Electrode and NanoAu Carrier Amplified Signal Strategy for Attomolar Mercury Detection Yi Zhang†,‡, Guang Ming Zeng*,†,‡, Lin Tang*,†,‡, Jun Chen†,‡, Yuan Zhu†,‡, Xiao Xiao He†,‡, Yan He†,‡

†

College of Environmental Science and Engineering, Hunan University, Changsha,

Hunan 410082, P.R.China ‡

Key Laboratory of Environmental Biology and Pollution Control of Ministry of

Education, Hunan University, , Changsha, Hunan 410082, P.R.China

Corresponding author. ∗

E-mail:

[email protected]

(G.M.

Zeng),

[email protected]

[email protected] (Y. Zhang); Tel: +86-731-88822754; fax: +86-731-88823701.

1

ACS Paragon Plus Environment

(L.

Tang),


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Abstract An electrochemical sensor was developed for attomolar Hg2+ detection. Three single-stranded DNA probes were rationally designed for selective and sensitive detection of the target, which combined T-Hg2+-T coordination chemistry and the characteristic of convenient modification of electrochemical signal indicator. Graphene and nanoAu were successively electrodeposited on a glass carbon electrode surface to improve the electrode conductivity and functionalize with the 10-mer thymine-rich DNA probe (P1). NanoAu carriers functionalised with 29-mer guanine-rich DNA probe (P3) labeled methyl blue (MB-nanoAu-P3s) were used to further strengthen signal response. In the presence of Hg2+, a T-T mismatched dsDNA would occur between P1 and a 22-mer thymine-rich DNA probe (P2) on the electrode surface due to T-Hg2+-T coordination chemistry. Followed by adding the MB-nanoAu-P3s for hybridization with P2, square wave voltammetry was executed. Under optimal conditions, Hg2+ could be detected in the range from 1.0 aM to 100 nM with a detection limit of 0.001 aM. Selectivity measurements reveal that the sensor is specific for Hg2+ even with interference by high concentrations of other metal ions. Three different environmental samples were analyzed by the sensor and the results were compared with that from an atomic fluorescence spectrometry. The developed sensor was demonstrated to achieve excellent detectability. It may be applied to development of ultrasensitive detection strategies.

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Mercury is a heavy metal known for its severe effects on human health and the environment owning to its neurotoxicity and physiological toxicity.1,2 The annual total global mercury emission from nature and human activities is approximately 7500 tons per year.3 Increasing efforts are being made to understand mercury distribution and pollution to address mercury exposure and improve public health.4,5 Thus, many methods have been developed for mercury detection, including inductively coupled plasma mass spectrometry,6 atomic absorption/emission

spectroscop,7

cold

vapor

atomic

fluorescence

spectrometry,8

high-performance liquid chromatography,9 ion chromatography,10 and sensing strategy.11-21 Although no one method has proven to be superior, sensing strategy is often reported due to its tremendous versatility. It negates the use of sophisticated instrumentation and complicated sample preparation process. In the last decade, mercury-sensing strategies based on thymine-Hg2+-thymine (T-Hg2+-T) coordination chemistry and the resulting Hg2+-stabilized hybridization of oligonucleotides with T-T mismatches have been developed rapidly. Indeed, since the specific recognition ability of Hg2+ using T-T base pairs in DNA duplexes was observed,11 several strategies for the detection of Hg2+, based upon electrochemistry,12,13 fluorescence,14,15 colorimetry,16,17 photoelectrochemistry,18,19 surface plasmon resonance,20 and surface resonance Raman scattering21 have been developed. The application of fluorophores,22 oxidoreductases,23 DNAzymes,24 conjugated polymers,25 intercalators,26 quantum dots,27 and nanomaterials28 diversified the sensors fabrication and also substantially enhanced the detectability of sensors. Among these techniques, colorimetry is an only visual technique that lacks sensitivity. Fluorescent techniques are sensitive but fluorescent indicators are prone to interference and they can yield false positive results. Although the problem of false positive results was somewhat circumvented by the development of quantum dots, there is still a need to demonstrate that quantum dots are not health threatening. 29 Electrochemistry, by contrast, is relatively convenient and stable, and has showed high sensitivity and excellent flexibility.26 In electrochemical sensing techniques, the development of nanomaterials30-32 and signal amplification strategies33,34, has significantly improved their detectability.35,36 Graphene, a one-atom-thick, two-dimensional material, has attracted great attentions because of high electron mobility and low electrical resistivity. It is known to significantly improve electrical 3



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conductivity of electronic components. It has also been used in many sensing strategies to achieve target detection with high sensitivity.37 Many methods e.g. micro mechanical stripping, chemical disoxidation of graphene oxide, chemical vapor deposition and etc., have been developed for the preparation of graphene.38 However, before further use, the produced graphene must be functionalized with chemical groups to improve its applicability because graphene is hydrophobic. The preparation processes involved a series of chemical reactions and require cumbersome control of reactions conditions.39 Compared with the above mentioned methods, the electrodeposition method is more convenient and environmentally friendly. In electrodeposition process, graphene oxide in a dispersion can be reduced to graphene, which can then be directly deposited on an electrode surface.40 Very often, gold nanoparticles are electrodeposited to form a graphene-gold nanoparticles modified electrode for detection of biomolecules. Graphene is an inert material that has no significant effect on the bioactivity of biomolecules,37 while gold nanoparticle has been reported a lot as a biocompatible material to biomolecules.41,42 Such an electrode shows an excellent affinity and biocompatibility to biomolecules.43,44 The electrodeposited graphene-gold nanoparticles layer (graphene-EAu) significantly enhances the conductibility of the electrode by acting as an electron mediator. In addition, nanoAu carrier amplified signal strategy was designed for fabrication the mercury sensor. A proper signal amplification strategy not only provides convenient and steady bridge between carrier and biomolecules, but also carries more response signal indicators into detection system, which maximizes the signal source, and then would be beneficial to obtain a favorable result.33 Methyl blue, a commonly used electrochemical signal indicator, can attach to a guanine-rich single stranded DNA due to the affinity of MB towards guanine.45 It facilitates the nanoAu carrier signal amplification for an electrochemical sensor using DNA strands. Herein, we reported that thymine-Hg2+-thymine interaction can be activated for Hg2+ detection with a detection limit of 0.001 aM. The result was mainly attributed to the improvement of electrical conductivity of working electrode by the graphene-EAu and the advanced signal amplification by nanoAu carrier strategy. Although the exciting detectability was obtained, the sensor construction was not complicated. Furthermore, to investigate the 4


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mercury detection of the sensor in environmental samples, atomic fluorescence spectrometry (AFS) was performed as a comparison test.

Experimental Section Materials and Apparatus. Mercury nitrate and 6-mercapto-1-hexanol (MCH) were purchased

from

Sigma-Aldrich

Chemical

Co.

Hydrochloroauric

acid

and

tris(2-carboxyethyl)phosphine (TCEP) were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Graphite oxide was purchased from XF Nano Inc. (Nanjing, China). Oligonucleotides were synthesized by Sangon Biotech. Co., Ltd. (Shanghai, China). All chemicals were of analytical grade. In our work, a Tris-HCl buffer (10 mM Tris adjusted pH with 10 mM HCl) and a phosphate buffer saline (PBS; 10 mM) were used. The synthesized oligonucleotides were provided by Sangon Biotech. Co., Ltd. (Shanghai, China). The sequences

were

as

5’-SH-(CH2)6-GTGTTTCTCA-3’

follows:

5’-GTGGAGAGAGGGTGTGTTTCAC-3’

(P2);

(P1);

5’-AAAAAGGGAGGGAGGGAGGG

TCTCTCCAC-3’ (P3). Probe 1 (P1) was dissolved in Tris-HCl buffer containing 0.1mM TCEP, 1mM EDTA and 1.0 M KCl. Probe 2 (P2) and Probe 3 (P3) were dissolved in Tris-HCl buffer containing 1.0 M KCl. They were kept at –20oC for further use. Electrochemical measurements were carried out on a CHI760D electrochemistry system (Chenhua Instrument, Shanghai, China). The three-electrode system used in this work consisted of a glass carbon electrode (GCE, 3 mm in diameter) as working electrode, a saturated calomel electrode (SCE) as reference electrode and a Pt foil auxiliary electrode. Scanning electron microscopy (SEM) of the morphology of the electrode surface was obtained using a JSM-6700F field emission scanning electron microscope (JEOL Ltd., Japan). A Sigma 4K15 laboratory centrifuge, a Sigma 1-14 Microcentrifuge (Sigma, Germany), an AFS-9700 atomic fluorescence spectrophotometer (Kechuang Haiguang Instrument, Beijing, China) and a Model CS501-SP thermostat (Huida Instrument, Chongqing, China) were used in the assay. All work was performed at room temperature (25oC) unless otherwise mentioned. Sensor Fabrication. First, graphene and Au were electrodeposited on the GCE surface that was treated by H2SO4, before a thiolated P1 was self-assembled on the electrode. Briefly, the

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graphite oxide powder was added in a PBS (pH 9.18), and was exfoliated by ultrasonication to form a 1.0 mg mL−1 graphene oxide colloidal dispersion. Then, cyclic voltammetric reduction was performed in the dispersion between –1.5 and 0.5 V at 10 mV s-1 with a magnetic stirring and N2 bubbling at 4oC. Subsequently Au was electrodeposited on the graphene modified GCE using chronoamperometry in 1% (w/w) HAuCl4 solution containing 0.5 M perchloric acid at a potential of 0.18 V. Next, following self-assembly 1 µM of P1 on the GCE-graphene-EAu, the electrode was treated with 1 mM of MCH for 30 min. Preparation of methyl blue Labeled NanoAu-Carrier Signal Probes. First, nanoAu carriers were prepared using the citrate and borohydride reduction method.46 Briefly, water (36.8 mL), HAuCl4 solution (1 mL, 0.01 M), and trisodium citrate solution (1 mL, 0.01 M) were mixed, then icecold aqueous sodium borohydride solution (1.2 mL, 0.1 M) was added all at once with stirring. The solution was placed in darkness for 3 hours to degrade any remaining borohydride by reaction with water. Next, the signal probes (P3) of 2.5 µM were self-assembled onto the nanoAu carrier (nanoAu-P3s). The nanoAu-P3s mixed with PBS containing 3 M NaCl and 25 mM MB, which was centrifuged (12000 rpm) for 15 min at 4 oC to remove the supernatant. After repeating the MB treatment and centrifugation one more time, the MB-nanoAu-P3s was redissolved in PBS containing 3 M NaCl to obtain a final concentration of 20 nM. Detection. The GCE-graphene-EAu-P1 was placed in 200 µL of Hg2+ solution containing 1 µM P2 for 30 min, and then washed using Tris-HCl (pH 7.4) containing 1.0 M KCl. This was followed by application of 30 µL of MB-nanoAu-P3s on the electrode surface to promote hybridization for 30 min. After being washed using Tris-HCl (pH 7.4) containing 1.0 M KCl, square wave voltammetry (SWV) was conducted at the electrode in 20 mL of Tris-HCl (pH 7.4) containing 10 mM KCl. The SWV was carried out between –0.7 and 0 V under pulse amplitude of 25 mV and frequency of 10 Hz, with a step potential of 4 mV. Optimisation experiments, detection limit and selectivity tests were next performed. In all optimisation experiments and selectivity measurement, 10 nM Hg2+ was used. Environmental Samples Analysis. Three different environmental samples, tap water, river water, and landfill leachate sample were used in this study. The river water was from the Xiangjiang river in Changsha, China, while the landfill leachate was obtained form municipal 6


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solid waste landfills in Changsha, China. They were centrifuged at 10000 rpm for 5 min and filtered to remove the suspension and solid impurities. The pH of the sample solutions was then adjusted to 7.4. The solutions were next spiked with different concentrations of Hg2+. Detection of Hg2+ was then conducted using the developed biosensor. Meanwhile, the same samples were filtrated via the 0.2-µm polycarbonate filter and analyzed by atomic fluorescence spectrometry (AFS) as a standard detection method for validation the sensor.

Result and Discussion Design of the Sensing Strategy. The sensing strategy is shown in Scheme 1. Graphene and Au were sequentially electrodeposited on a GCE surface, followed by the self-assembly of the thiol modified oligonucleotide capture probe, P1. This assembly was stabilised on the electrode by the thiol-Au affinity.47 After treatment with MCH for eliminating nonspecific binding on electrode surface, the GCE-graphene-EAu-P1 was allowed to interact with Hg2+ sample solution containing the oligonucleotide target probe, P2. Following the addition of the MB labeled signal probe, P3, electrochemical detection was carried out. The three probes used here were rationally designed to ensure the selectivity and sensitivity of the mercury sensor. P1 and P2 are incomplete complementary strands. In the absence of Hg2+, P1 and P2 cannot form a double-stranded-DNA (dsDNA) on the electrode surface. Meanwhile, there is also no signal response from the MB-labeled P3. In the presence of Hg2+, however, a T-T mismatched dsDNA would occur between P1 and P2 on the electrode surface due to T-Hg2+-T coordination chemistry. At this point, MB labeled nanoAu-carrier signal probes (MB-nanoAu-P3s) joined in the detection system and offered the available detection signal. P2 and P3 can form dsDNA by hybridization because partial sequences in P2 and P3 are completely complementary. P3s were bound onto the nanoAu-carrier with the help of poly adenine, which serves as a binder for the conjugate of DNA and nanoAu and can control orientation without modifications.48 Moreover, attributed to the affinity of MB towards guanine,45 sufficient MBs were loaded on the P3s, to which polyguanine was attached. Additionally, the nanoAu carrier amplified signal strategy can offer more response signal source in the limited reaction sites of the reaction system, which is benefit for the sensitivity of the sensor. Thus, the sensor design would display a satisfactory detection capability in

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theory. Characteristic of Electrode. Graphene and Au were electrodeposited on a GCE. Typical cyclic voltammograms (CV) of graphene oxide electrolysis on the GCE, depicted in Figure 1a, show that the voltammetric current increased with successive potential scans, indicating that deposition of conducting graphene on the GCE took place. This result was also supported by the analysis of the scanning electron microscope (SEM), which showed a veil-like film coated on the GCE surface (Figure 1b). To further improve the microenvironment on the electrode surface for immobilizing DNA probes, Au nanoparticles were electrodeposited on the GCE-graphene surface for 50s getting the largest peak current response (Figure 1c). An appropriate amount of electrodeposited Au would be advantageous to the graphene-EAu system, i.e. excessive EAu would weaken the effect of graphene and get a similar effect of a gold electrode, which covered the graphene film and hindered the electron transport in graphene. Conversely, too little EAu would not provide sufficient reaction sites for binding DNA probes, and would result in inadequate response. In the SEM, the scattered electrodeposited Au particles can be clearly seen (Figure 1 d), and the veil-like graphene is dispersed among these Au particles. Optimization of Experimental Conditions. A series of experiments was performed to optimize the conditions with acceptable signal response. The effect of pH condition, treatment time of MB to nanoAu-P3s, the amount of MB-nanoAu-P3s, and reacting time with Hg2+ ion were investigated. The effect of temperature was not considered because the Hg2+ sensor was only designed to be applied at room temperature. It is important to study the effect of pH of environmental samples on the Hg2+ sensor response. In Figure 2a, the maximum of peak current response in SWV appeared in pH 7.0 to 7.5, and its more than 70% peak current response was reached in pH 6.0 to 8.0, which showed the sensor had a certain pH buffer capacity. Additionally, the peak current exhibited a positive correlation with the treatment time of MB to nanoAu-P3s over a period of 30 min (15 min×2). The largest peak current appeared in the treatment of MB for 30 min, however, the current growth between the treatment time of MB 20 to 30 min was about a quarter of that between the treatment time of MB 10 to 20 min (Figure 2b). From the current response and time cost point of view, exceeding 30 min of the treatment with MB would drive down the efficiency of 8


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mercury detection. Therefore, 30 min of the treatment time of MB to nanoAu-P3s was chosen in subsequent tests. In the test the optimum amount of MB-nanoAu-P3s (Figure 2c), although 25 nM of MB-nanoAu-P3s contributed the largest peak current, 20 nM of that got a similar result. This means 20 nM of MB-nanoAu-P3s was the most cost-effective amount for the sensor. While in Figure 2d, with the increasing of reacting time with Hg2+ the current response had an upturn and became stable in the reacting time of 30 min, therefore, the reacting time of 30 min with Hg2+ was as the optimization result. Effects of Graphene-EAu and the Signal Amplification Strategy. To investigate the effects of graphene-EAu and the signal amplification strategy, the graphene-EAu modified electrodes and EAu modified electrodes with/without using the nanoAu carrier amplified signal strategy (nanoAu-P3) were used to test the same samples and the results are shown in Figure 3. The largest current response was obtained at a nanoAu-P3 modified graphene-EAu electrode, while the lowest response observed at an EAu only modified electrode. Compared to the peak current response of graphene-EAu modified electrodes, there was a more than 70% decrease in the current response observed at EAu modified electrodes. Although a relatively small effect to the detection system, the nearly 30% improvement of response signal is attributed to the nanoAu-P3 strategy. Therefore, the Ge-EAu modified electrodes with nanoAu carrier amplified signal strategy could enhance the response signal intensity several times. Detectability of the Sensor. To evaluate the detectability of the sensor, various concentrations of Hg2+ from one stock solution were tested. As mentioned above, in the absence of Hg2+, no response signal was detected. While in the presence of Hg2+, the response signals were sensitive to Hg2+and increased along with the increase of Hg2+ concentrations (Figure 4a). A linear correlation between peak current and the logarithm of Hg2+ concentrations was observed in the range from 1.0 aM to 100 nM with a detection limit of 0.001 aM (the mean value of background signals plus three times standard deviation of background signals, Figure 4b). Each point of the calibration was done in triplicates, and the maximum relative standard deviation was 4.49%, which guaranteed the precision of the proposed sensor. The detection limit is superior to the above mentioned sensors and others based on graphene for mercury detection, such as a fluorescence sensor using graphene quantum dot with a detection limit of 0.12 µM,49 a field-effect transistor sensor based on a 9



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transparent substrate deposited graphene with a detection limit of 10 pM,50 and an electrochemical sensor based on chitosan immobilized graphene and nanoAu as signal reporter with a detection limit of 0.06 nM.51 The result was mainly attributed to the improvement of electrical conductivity of working electrode by the graphene-EAu and the advanced signal amplification by nanoAu carrier strategy. Selectivity of the Sensor. To examine the selectivity of the sensor for Hg2+, some samples containing environmentally relevant metal ions were tested, including K+, Ba2+, Ca2+, Cd2+, Co2+, Cr2+, Cu2+, Mg2+, Mn2+, Ni2+, Pb2+, Zn2+, Al3+, Fe3+ at a concentration of 500 nM, and their mixtures (Figure 5). The response from these environmentally relevant metal ions was less than 5% compared with that of 10 nM Hg2+. Apparently, the interference of these environmentally relevant metal ions to the sensor is negligible. Analysis of Hg2+ in Samples. With excellent sensitivity and selectivity in buffer solution, the mercuric sensor was further tested in environmental samples. The tap water, river water, and landfill leachate sample were spiked with Hg2+ ion and analyzed by the proposed sensor and AFS. The detection results of the two methods were presented in Table 1. When Hg2+ ion concentration was greater than 0.05 nM, the detection results of the two methods were basically the same. However, when Hg2+ ion concentration was lower than 0.05 nM, it was undetectable to AFS, but not to the sensor. Additionally, the detection results of the two methods were compared by paired t-test. According to

t=

d Sd

(1)

n

where d is mean deviation, Sd is standard deviation, n is pairing number. Calculation shows that t is 1.2201 and is less than the given t critical value, i.e. the mercury sensor is of reliability as AFS for Hg2+ detection. The Above results indicated the potential of the sensor as an ultrasensitive and reliable analysis method of Hg2+ in the environmental samples.

Conclusions In conclusion, an ultrasensitive method based on nanomaterials and the relative functionalized strategies for Hg2+ detection with a detection limit of 0.001 aM was developed. The detection sensitivity was enhanced by several orders of magnitude from what had been

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reported, which is much lower than the EPA limit of Hg2+ ions in drinking water (10 nM), and would meet the demand of high precision detection. Three components of the sensor contribute to its significant improvement of detection performance: 1) graphene and Au were conveniently electrodeposited onto the electrode surface for improving electrode performance and functionalization without any modification that might hinder the conductibility of electrode; 2) the three rationally designed oligonucleotide probes gave full play to the effect of T-Hg2+-T coordination chemistry to ensure high selectivity and sensitivity of the detection system; 3) the nanoAu carrier amplified signal strategy further strengthened the signal characterization capabilities and improved the detectability. Thus, the idea of sensor construction could obtain the exciting detectability and deserves to be promoted for developing ultrasensitive detection strategies.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 51039001, 51222805 and 51378190), the Young Top-Notch Talent Support Program from Chinese Government (2012), the Program for Changjiang Scholars and Innovative Research Team in University (IRT-13R17), the fundamental Research Funds for the Central Universities, Hunan University and the Hunan Provincial Innovation Foundation For Postgraduate (CX2009B080).

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Tables TABLE 1. Mercury Concentration in Environmental Sample Determined by Mercuric Sensor and AFS

15



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TABLE 1. Mercury Concentration in Environmental Sample Determined by Mercuric Sensor and AFS mercury concentration (nM) samples added mercury sensor a AFS b deviation tap water

river water

landfill leachate a

10 0.5 0.05 10 0.02 0.001 10 100 0.005

10.0733±0.1726 0.4941±0.0253 0.0504±0.0013 10.1094±0.2096 0.0213±0.0012 0.0009±0.0004 9.9473±0.2785 100.9450±2.0834 0.0052±0.0004

10.1226±0.1451 0.513±0.0267 0.0526±0.0267 10.1832±0.2175 – – 10.2623±0.2936 103.4521±2.5272 –

0.0493 0.0189 0.0022 0.0738 – – 0.315 2.5071 –

An average of three replicate measurement. b An average of three replicate measurement.

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Figures Scheme 1. Sketch map of the sensing strategy for mercury detection.

Figure 1. Characterization of the modified electrode surface: a) cyclic voltammogram of electrodeposition graphene on a GCE by electrochemical reduction of 1.0 mg mL-1 graphene oxide in PBS (0.067 M, pH 9.18) at 10 mV s-1; b) SEM image of the graphene modified GCE; c) Square wave voltammogram of the graphene-EAu modified GCE with different time (10,20,30,40,50,60,100s) of electrodeposition of Au to 100 nM of Hg2+ ion in 20 mL of Tris containing 10 mM KCl (10mM, pH 7.4) between –0.7 and 0 V under pulse amplitude of 25 mV and frequency of 10 Hz with a step potential of 4 mV; d) SEM image of the graphene-EAu modified GCE.

Figure 2. Optimization of experimental conditions: a) effect of pH condition,; b) effect of reatment time of MB to nanoAu-P3s; c) amount of MB-nanoAu-P3s; d) effect of reacting time with Hg2+ ion. Under the conditions of 10 nM of Hg2+ and 1 µM of P2, the electrodes modified 1 µM of P1 were used to investigate the relative factors. Deviation for the mean of three replicate tests.

Figure 3. Effect of graphene-EAu and the signal amplification strategy for the mercuric sensor by square wave voltammetry to 100 nM of Hg2+ ion in 20 mL of Tris containing 10 mM KCl (10mM, pH 7.4) between –0.7 and 0 V under pulse amplitude of 25 mV and frequency of 10 Hz with a step potential of 4 mV.

Figure 4. Square wave voltammograms of mercuric sensor a) to various concentrations of Hg2+ ions in 20 mL of Tris containing 10 mM KCl (10mM, pH 7.4) between –0.7 and 0 V under pulse amplitude of 25 mV and frequency of 10 Hz with a step potential of 4 mV. And b) calibration plot of response current vs. logarithm of Hg2+ ion concentration between 1 aM to 100 nM. The vertical bars designate the standard deviation for the mean of three replicate tests.

17



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Figure 5. Interference test results using square wave voltammetry by mercuric sensor in 20 mL of Tris containing 10 mM KCl (10mM, pH 7.4). Under the optimal experimental conditions, 10 nM of Hg2+, 500 nM of K+, Ba2+, Ca2+, Cd2+, Co2+, Cr2+, Cu2+, Mg2+, Mn2+, Ni2+, Pb2+, Zn2+, Al3+, Fe3+, and their mixture containing 10 nM of Hg2+ were respectively measured. The vertical bars designate the standard deviation for the mean of three replicate tests.

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Scheme 1 GCE GCE-graphene GCE-graphene-EAu GCE-graphene-EAu-P1

MB-nanoAu-P3s

P2

Hg2+ + P2

MB-nanoAu-P3s G T

T C

G

T

GG A

C

T

TT T T C T A G C T G

GCE-graphene-EAu-P1-P2

G T G G A G A C T G T C TG A C C A C G G G AG G G A G A G G G G G AA AA AA AA AAA A AAAA

P1

P2 NanoAu

P3 MCH

graphene Hg2+


MB


Figure 1 5

a)

Current / 

0

-5

-10

-15

-20 -1.5

-1.0

-.5

0.0

.5

Potential / V 10

c)

8

Current / 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10 s 20 s 30 s 40 s 50 s 60 s 100 s

6

4

2

0 -.7

-.6

-.5

-.4

-.3

-.2

-.1

0.0

Potential / V


Page 20 of 25

Page 21 of 25

Figure 2 10

a)

b)

8

15x2 min 10x2 min 5x2 min

8

Current / 

Current response in SWV / 

10

6

4

6

4

2

0

2 5.5

6.0

6.5

7.0

7.5

8.0

8.5

-.7

9.0

-.6

-.5

pH

-.4

-.3

-.2

-.1

0.0

Potential / V

c)

8

Current response in SWV / A

10

Current / 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25 nM 20 nM 15 nM 10 nM

6

4

2

0

9

d)

8

7

6

5 -.7

-.6

-.5

-.4

-.3

-.2

-.1

0.0

15

20

Potential / V


25

30

Time /minute

35

40


Figure 3 10 Graphene-EAu--[nanoAu-P3] Graphene-EAu--[P3] EAu--[nanoAu-P3] EAu--[P3]

8

Current / 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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6

4

2

0 -.7

-.6

-.5

-.4

-.3

-.2

Potential / V


-.1

0.0

Page 23 of 25

Figure 4 10

10 M 100 nM 1 nM 10 pM 100 fM 1 fM 10 aM 0.1 aM 0.001 aM

a)

Current / 

8 6

1 M 10 nM 100 pM 1 pM 10 fM 100 aM 1 aM 0.01 aM

4 2 0 -.7

-.6

-.5

-.4

-.3

-.2

-.1

0.0

Potential / V 12

b)

10

Current / 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8 6

y=(0.4043±0.01)x+(4.7463±0.0649)

4

R2=0.9839

2 0 -4

-2

0

2

4

6

8

10

Log(Hg2+ concentration) / aM


12

14


Figure 5 10

8



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6

4

2

0 K+ Ba2+ Ca2+ Cd2+Co2+Cr2+ Cu2+ Mg2+Mn2+ Ni2+ Pb2+ Zn2+ Al3+ Fe3+ Hg2+Hg2++mix

Metal ions


Page 24 of 25

Page 25 of 25

For TOC Only

G T

A C

G

T C

G

T

GG A

C

T

TT T T C T G T

G A G A C T GG T G T C TG AC C AC G GG AG G G A G G A G G G G AA AA AA AA AAA A AAAA

10

10 M 100 nM 1 nM 10 pM 100 fM 1 fM 10 aM 0.1 aM 0.001 aM

8

Current / 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


6

1 M 10 nM 100 pM 1 pM 10 fM 100 aM 1 aM 0.01 aM

4 2 0

Graphene-EAu modified GCE

NanoAu carrier signal

amplification

-.7

-.6

-.5

-.4

-.3

-.2

-.1

0.0

Potential / V


.1

Electrochemical Sensor Based on Electrodeposited Graphene-Au

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