Copper-Nitrogen Doped Graphene Hybrid as an Electrochemical

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Copper-Nitrogen Doped Graphene Hybrid as an Electrochemical Sensing Platform for Distinguishing DNA Bases Shu-Wen Sun, Hai-Ling Liu, Yue Zhou, Feng-Bin Wang, and Xing-Hua Xia Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02520 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 24, 2017

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Copper-Nitrogen Doped Graphene Hybrid as an Electrochemical Sensing Platform for Distinguishing DNA Bases Shu-Wen Suna, b, Hai-Ling Liua, Yue Zhoua, Feng-Bin Wanga, Xing-Hua Xiaa* a State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, China. b Department of Applied Chemistry, Yuncheng University, Yuncheng, 044000, China.

ABSTRACT: An electrochemical sensor using ultralight and porous copper-nitrogen doped graphene (CuNRGO) nanocomposite as the electrocatalyst has been constructed to simultaneously determine DNA bases such as guanine (G) and cytosine (C), adenine (A) and thymine (T). The nanocomposite is synthesized by thermally annealing an ice templated structure of graphene oxide (GO) and Cu(phen)2. Due to the unique structure and the presence of Cu2+-N active sites, the CuNRGO exhibits outstanding electrocatalytic activity toward the oxidation of free DNA bases. After optimizing the experimental conditions, the CuNRGO based electrochemical sensor shows a good linear responses for G, A, T and C bases in the concentration range of 0.132-6.62 μM, 0.37-5.18 μM, 198.25551 μM and 270.0-1575 μM, respectively. The results demonstrate that CuNRGO is a promising electrocatalyst for electrochemical sensing devices.

Deoxyribonucleic acids (DNAs) are important biomolecules which have three-dimensional structures containing two polynucleotide chains. Two complementary base pairs: Guanine (G) and cytosine (C), adenine (A) and thymine (T) are coiled around the chains and connected mainly with the stability of doublestranded DNA, plenty of genetic information and DNA replication.1 The abnormal changes of bases are considered as deficiency and mutation of the immunity and may induce various diseases, such as cystic fibrosis, Parkonson’s disease, diabetes, Alzheimer’s disease and various cancers.2 As an imperative of early detection and treatment of diagnose, various studies on single nucleotide polymorphism (SNP) has been carried out to provide detailed information about single base mismatch.3-5 Therefore, the development of accurate and sensitive methods for individual and/or simultaneous determination of DNA bases is of great importance in bioscience and clinical diagnosis. In recent years, various approaches to the detection and quantification of G and A bases have been proposed including highperformance liquid chromatography,6 chemiluminescence method,7,8 spectroscopic methods,9 capillary electrophoresis10 and mass spectrometry.11 As is known, these methods have limitation in simultaneously determination of four bases without separation processes. Thus, difficulty exists in determination of the four bases using the above mentioned methods since the required pretreatments are often time-consuming and will inevitably introduce determination errors. Electrochemical methods are of various advantages including fast performance, high sensitivity, easy miniaturization, and good selectivity if coupled with surface property modifications of the electrodes. They have been proved to be excellent alternatives to directly detect targets without sample pretreatment12. It has been reported that G and A can be detected on conventional glassy carbon (GC) electrode. However, direct detection of T or C on GC electrode is difficult since the extremely positive oxidation potential of pyrimidine exceeds the narrowed electrochemical potential

window of the GC electrode. In addition, the pure GC electrode surface will be easily poisoned due to its high affinity to oligonucleotides, which leads to a decline in both sensitivity and reproducibility. Modification of the GC electrode with carbonbased composites has been reported as alternative measure to overcome the above mentioned problems in determination of DNA bases.13-18 Niwa et al. reported that the nanohybrid carbon film with superior stability, extended electrochemical potential window, and good catalytic activity as compared to the pure GC electrode could be used to sensitively and selectively detect SNPs in oligonucleotides15 including the sequence from codon 248 of the p53 gene in pH = 5 acetate solution, although this catalyst was not yet suitable for determining longer DNA fragments. Electrochemical sensors with alternative nanomaterials as the catalysts have shown various advantages, however they could be used to detect only individual or simultaneous determination of one or two purine bases.14,18-20 Therefore, an enormous challenge remains to establish direct oxidation electrochemical assays for simultaneous detection of four free DNA bases. As one kind of carbon allotropes, graphene has attracted extreme attentions due to its unique structure and electronic properties and has been extensively applied to the construction of nanoelectronics, energy storage devices, and biosensors. Importance has been demonstrated in the construction of electrochemical assays for direct analysis of DNA21 and SNP.22,23 In the case of SNP electrochemical assay, several hybridization processes, however, are required in order to precisely detect single base mismatch, which restrains its practical application in clinical diagnosis. Recently, we synthesized a bio-inspired copper nanocomposite prepared by thermal decomposition of Cu2+-1,10-phenathroline complexes (Cu(phen)2) and graphene oxide (GO) precursors, which showed excellent catalyst toward the oxygen reduction/evolution reactions and glucose oxidation in alkaline

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solutions.24,25 In this work, we demonstrate that the copper-nitrogen doped graphene (CuNRGO) nanocomposite shows excellent electrocatalytic activity toward the oxidation of four free DNA bases. To demonstrate the importance of high surface area and highly active sites in the electrochemical determination of DNA bases, ultralight and porous CuNRGO was synthesized with improved approach by thermal annealing an ice-template shaped structure of graphene oxide (GO) and Cu(phen)2. After characterization of the catalyst, systematical investigation on the electrochemical oxidation of four free DNA bases on the CuNRGO modified electrode was performed. Results show that the CuNRGO catalyst exhibits good electrocatalytic activity toward the oxidation of DNA bases with significant peak potential separations, and thus simultaneous determination of four DNA bases can be achieved in mixtures without any separation pretreatment. EXPERIMENTAL SECTION Reagents and Apparatus. Guanine, adenine, thymine and cytosine with high purity were purchased from Bio Basic Inc. (Shanghai, China). Deoxyribonucleic acid sodium salt from calf thymus (DNA) was obtained from Sigma Aldrich. Graphite powder (99.9995% purity, 100 mesh, briquetting grade, mesh) was purchased from Alfa Aesar. Cu(Ac)2·H2O and 1,10-phenanthroline (Cl2H8N2) were purchased from Sinopharm Chemical Reagent Co., Ltd., (Shanghai, China). All of the other chemicals such as H3PO4, NaOH, N,N-dimethylformamide (DMF), Na2HPO4 and NaH2PO4 from Nanjing Chemical Reagent CO. Ltd. were of analytical grade and used as received. Phosphate buffer solutions (PBS) with different pH values were prepared by mixing stock solution of 0.2 M Na2HPO4 and 0.2 M NaH2PO4. All solutions were freshly prepared with Millipore water having a resistivity of 18.2 MΩ (Purelab Classic Corp., USA). The morphology of samples was imaged on a field-emission scanning electron microscope (FE-SEM, S-4800, Hitachi, Japan) at an accelerating voltage of 10 kV. Transmission electron microscopic and HRTEM images of the samples on Cu-grids were obtained using a transmission electron microscopy (TEM, JEM-200CX, Japan) and a Hitachi-2100 TEM facility with a 200 kV accelerating voltage, respectively. An X-ray powder diffractometer (XRD, Shimadzu, X-6000, Cu KR radiation) was used to determine the phase purity and crystallization degree. Room temperature Raman spectra were recorded using a Renishaw InVia microRaman system with an excitation wavelength of 514 nm. XPS spectra were obtained on a PHI 5000 Versa Probe (Japan). Curve fitting of the high resolution N1s spectra was performed using a ~20% Lorentzian-Gaussian peak shape. The specific surface area and pore size distribution of the samples were calculated by Brunauer Emmett Teller (BET) analysis of nitrogen adsorption and desorption isotherm (ASAP2020, Micromeritics, USA). Synthesis of CuNRGO and similar contrast materials. Graphene oxide was synthesized by the modified Hummers method.26 The Cu(phen)2 precursor was synthesized by heating a mixture solution of Cu(Ac)2 (0.5 mmol in 30 ml dimethyl formamide) and 1,10-phenanthroline (1 mmol in 70 ml CH2Cl2) at 35 oC for 8 h.27 After the solvents of the reaction solution were removed by rotary evaporation at 45 oC, solid product was washed using acetone and CH2Cl2 until turning into emerald. A mixed solution of 10 mg Cu(phen)2, 10 mg GO and 15 ml water was frozen with liquid N2 after 30 min ultrasonication and then treated by freeze-drying for 40 h, resulting in a spongy solid.

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Pyrolysis of the spongy solid at 900 oC in Ar atmosphere for 1 h resulted in a fluffy composite. To exclude any unstable phases in the resulting material, a treatment with 0.5 M H2SO4 at 80 oC for 2 h was carried out. Reduced graphene oxide (RGO) was also synthesized under the same conditions just without Cu(phen)2. For control experiments, the Cu species in CuNRGO was removed by acid treatment of CuNRGO with an aqueous solution of 1.3 M HNO3 at 80 °C for 2 h. The resulting product is denoted as CuNRGO-HNO3. Then, the CuNRGOHNO3 sample was mixed with Cu(Ac)2 (80 mM) in DMF at room temperature for 12 h to re-attach Cu2+ ions to the graphene structure, afterward washed sequentially with ethanol and dried at room temperature. The product is denoted as CuNRGOHNO3-Cu2+. Preparation of the modified electrodes for electrochemical measurements. Prior to use, a glassy carbon electrode (GCE, Ф=3 mm) was polished with 1, 0.3, and 0.05 mm gamma alumina powders. After the polished electrode was washed with ethanol and water in an ultrasonic bath which remove the alumina residues, it was dried with N2 flow. The pretreated GCE was modified with 2 μL of 1.5 mg mL-1 of CuNRGO suspensions dispersed in DMF and dried in air. After the modification, the obtained electrode is donated as CuNRGO/GCE. The same amount of individual RGO, CuNRGO-HNO3 and CuNRGOHNO3-Cu2+ was dispersed in the same volume of DMF, and 2 μL of each dispersion was modified on the pretreated GCE and are donated as RGO/GCE, CuNRGO-HNO3/GCE, and CuNRGO-HNO3-Cu2+/GCE, respectively. All the modified electrodes were immersed in 0.1 M pH 7.4 PBS for half an hour before use and stored in air at room temperature when not in use. Differential pulse voltammetry (DPV) and cyclic voltammograms (CVs) were performed using a three-electrode configuration with a CHI 830B electrochemical workstation (CHI Instruments, Shanghai Chenhua Instrument Corporation, China). GCE or modified GCE was used as the working electrode, a Pt wire as the counter electrode, and an Ag/AgCl electrode (saturated KCl) as the reference. In collecting DPV profiles, the following parameters were used: increment = 4 mV, amplitude = 50 mV, pulse width = 0.2 s, pulse period = 0.5 s and sampling width = 0.0167 s. The electrochemical impedance spectroscopy (EIS) was carried out on an Autolab PGSTAT12 (Netherlands) in the frequency range from 1 Hz to 100 kHz in 5 mM Fe(CN)63/4+ 0.1 M KCl. RESULTS AND DISCUSSION Characterization of CuNRGO. After pyrolysis of the ice shaped Cu(phen)2 and GO mixture, the product was purified with sulfuric acid to remove any unstable phases. The formation mechanism refers to our reported work.24 As shown by the photo in Figure 1a, the resulting product CuNRGO with black color is very light and easy to flutter in the air, which could be due to the presence of abundant pores formed during the ice sublimation process. The SEM image (Figure 1b) shows that CuNRGO has a sheet-like structure involves wrinkles and folding details, displaying the typical features of graphene. Detailed TEM examination exhibits that CuNRGO has wrinkled thin layer structure (Figure 1c) and copper nanoparticles as dark dots are scattered on graphene. High resolution TEM image displays the lattice fringes spacing of ~0.202 nm for copper nanoparticles (Figure 1d). Nitrogen adsorption/desorption isotherms of

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be

Figure 1. (a) Photo of the porous CuNRGO sample on a pan paper; (b) SEM, (c) TEM and (d) High resolution TEM images of CuNRGO; (e) N2 adsorption/desorption isotherms of CuNRGO; (f) Pore size distribution of CuNRGO. CuNRGO exhibit the type IV features (Figure 1e). The corresponding Brunauer-Emmett-Teller (BET) analysis shows that the surface area of the CuNRGO is 184.8m2 g-1, which is much larger than 5.8 m2 g-1 for the sample prepared without ice template in control experiment. Figure 1f shows that the average pore size is near 2.5 nm. The significantly increased surface area of CuNRGO would be important for effective mass transport and increased surface active sites for electrocatalysis as reported for nitrogen doped graphene (NRGO).27 As shown by the XRD characterizations (Figure 2a), CuNRGO shows a characteristic band at 26.2° , which is near to the 002 reflection of natural graphite, demonstrating the good crystallinity of graphene.28 The interlayer distance of CuNRGO calculated using the Bragg's equation is 3.42 Å, approximately to the interlayer distance of RGO (3.49 Å). This demonstrates that the ice-template method will not significantly affect the interlayer spacing of the graphene structure. Beside the formation of graphene structure, a sharp diffraction peak at ~43° for CuNRGO in Figure 2b appears, which can be assigned to the Cu (111) crystal face of 2θ = 43° .29 This band is very small and has no significant changes for the CuNRGO sample washed by H2SO4, indicating that the Cu NPs is still remained on the graphene surface although the signal is not big enough for detection. Raman scattering spectra were measured with a laser excitation wavelength of 514 nm. As shown in Figure 2c and 2d, RGO displays the typical D band at 1370 cm-1 reflecting the breathing mode of κ-point photons of A1g symmetry and the existence of many defects, which can be attributed to harsh oxidation of graphite and doping elements in the carbon hexagonal lattice. While the G peak at 1596 cm-1 represents the E2g phonon of sp2 C atoms in graphene network. Compared to the spectrum of high quality single-layer graphene,30 the RGO exhibits a broader and weak 2D peak at about 2696 cm-1, implying that thermal annealing process generates few-layer graphene structures which are retained after nitrogen doping. This could also

the

main

Figure 2. (a) XRD patterns of GO, RGO, and CuNRGO; (b) Full XRD patterns of CuNRGO with and without treatment using H2SO4; (c) Raman spectra of GO, RGO, Cu(phen)2, and CuNRGO; (d) Raman spectra of GO, RGO, and CuNRGO. reason that nitrogen doping does not decrease the electrical conductivity of graphene. Characterization with X-ray photoelectron spectroscopy (XPS) was performed to analyze the composition of CuNRGO. The full range XPS spectrum (Figure S1a) of CuNRGO clearly shows the existence of carbon (C), nitrogen (N), oxygen (O) and copper (Cu) with the atomic percentages of 79.49%, 4.17%, 16.08% and 0.26% (Table S-1), respectively. The corresponding peaks for C1s, N1s, and O1s are centered at 284.5 eV, 400.9 eV, 531.7 eV, respectively. According to our earlier work,24 Cu2+-N-C structure exists in the frame. While peaks of Cu0 species and Cu2+ ions connected to the N atoms cannot be clearly found since their amount is very low. After the subtraction of a Shirley background, the C1s peak can be deconvoluted into three sub-peaks at 284.5, 285.8 and 287 eV (Figure S1b) by fitting a function of Lorentzian-Gaussian, which are assigned to highly oriented pyrolytic graphite (HOPG) connected with sp2 hybridization C=C, C=N and C-N bonds, respectively. After fitting based on Shirley algorithm, high resolution scans for N1s peak can be fitted into three components (Figure S1c). The two bands located at 398.6 eV and 399.9 eV are ascribed to the “pyridinic” and “pyrrolic” nitrogen configurations, respectively. The band at 401.1 eV is assigned to the N atoms bounded with three carbon atoms, i.e., graphitic” N atoms.31 From the integral area of the three kinds of nitrogen configurations, the “graphitic” N atoms percentage is dominant. A small O1s signal at 531.7 eV is due to the presence of minor oxygenated groups. The main signal at higher binding energy of 934.6 eV (Figure S1d) is originated from Cu2+ ions in Cu2+-N bond.24 The above results show that Cu2+-N is present in the catalyst and bounded with C in the graphene framework, but not simply absorbs on the surface. The band at 952.2 eV for Cu atom is trivial and almost embedded in the noise. The electrochemical impedance spectra (EIS) of the modified electrodes using redox probe Fe(CN)63−/4− are presented in Figure S2. The EIS consists of a semicircular and a linear parts. The diameter of the semicircle at high frequency region is equivalent to the electron transfer resistance (Ret) of the electrochemical probe.22 By fitting the data with Randles equivalent circuit (inset of Figure S2), the electron transfer resistance of

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the probe is 90.7Ω and 70.8 Ω at the GCE and RGO/GCE, respectively. For the CuNRGO/GCE, the Nyquist plot shows almost a linear, demonstrating relatively lower electron transfer resistance of the probe. This could be due to the positive charges carried by CuNRGO which attracts the negatively charged probe. The fastest Ret of CuNRGO shows its advantages for base electrocatalysis than the other two catalysts. Electrocatalysis of G, A, T and C at CuNRGO/GCE. The electrochemistry of a mixture containing G and A, T and C in PBS (pH = 7.4) was investigated at a bare GCE, RGO/GCE and CuNRGO/GCE. As expected, there does not exhibit any Faradaic features for the GCE in PBS base electrolyte (Figure

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ones on CuNRGO-HNO3. These results demonstrate that the Cu2+-N structure is indeed the active site for catalyzing the electrochemical oxidation of the DNA bases. However, it is noticed that the electrocatalytic activity of CuNRGO-HNO3-Cu2+ is not yet comparable to the original CuNRGO catalyst, demonstrating the importance of the Cu0 species connected to the Cu2+ ions in the electrocatalytic oxidation of four bases as suggested in our previous report.24,25 Effect of scan rate on the electrocatalytic oxidation of DNA bases. sCyclic voltammograms (CVs) of a CuNRGO/GCE in (pH = 7.4) PBS containing G, A, T, and C were recorded at various scan rates (Figure S4). From the CVs, it clear that the oxidation of each base is irreversible completely. The oxidation peak current (Ip) for G, A, T, and C is directly proportional to the scan rate in the studied ranges. The linear regression equations and correlation coefficients are: ( ) = 0.0476 ( / ) + 20.76( = 0.9996) I (μA) = 0.0328v(

/ ) + 4.134(r = 0.9994)

I (μA) = 0.0139v(mV/s) + 1.916(r = 0.9992) I (μA) = 0.1191v(mV/s) + 1.046(r = 0.9986)

Figure 3. DPVs of a mixture containing G (20 μg mL-1), A (20 μg mL-1), T (30 μg mL-1) and C (30 μg mL-1) in PBS 7.4 at GCE (curve b), RGO/GCE (curve c), and CuNRGO/GCE (curve d). For comparison, the DPVs of a GCE in PBS (pH = 7.4) is also presented (curve a). 3, curve a). The DPV of the four bases on bare GCE (Figure 3, curve b) shows two broad oxidation peaks at about 0.724 and 1.040 V due to the overlap of the electrocatalytic oxidation signals of the four bases. The four bases can be distinguished on RGO/GCE as revealed by the appearance of four oxidation peaks (Figure 3, curve c) at 0.596, 0.844, 1.056, and 1.232 V, respectively, which is in good agreement with the results reported previously.21 As compared to the RGO/GCE, the CuNRGO/GCE shows much better electrocatalytic activity toward the four bases as revealed by the lower oxidation overpotentials and higher oxidation currents for the four bases (Figure 3, curve d). The oxidation peaks for G, A, T, and C are negatively shifted to 0.592, 0.876, 1.056 and 1.208 V, respectively. The direct peak potential resolutions between G and A, A and T, T and C are 284, 180, 152 mV, respectively, which is large enough for their potential recognition and simultaneous detection. The excellent electrocatalytic activity of CuNRGO/GCE toward the oxidation of DNA bases could be attributed to the presence of Cu2+-N active sites. To confirm the special Cu2+-N structure as the active site for nucleobase oxidation, a control experiment using CuNRGOHNO3 as the catalyst was performed. The CuNRGO-HNO3 obtained by HNO3 treatment of CuNRGO should not consist of any copper species as we reported previously.24,25 The results are shown in Figure S3. As expected, the nitric acid leaching process degrades the electrocatalytic activity of the catalyst as revealed by the positive shift of the oxidation potential and significantly decreased oxidation current for the four DNA bases. If copper ions are re-coordinated to the CuNRGO-HNO3 catalyst forming CuNRGO-HNO3-Cu2+, partial recovery of the electrocatalytic activity of the catalyst is observed as shown by the increased oxidation currents and negative shifts of the oxidation potentials for the four DNA bases as compared to the

for G, A, T, and C, respectively. The results indicate that the electrode reactions of G, A, T, and C on CuNRGO/GCE are adsorption-controlled processes, which demonstrate that the current waves are due to the oxidation of the adsorbed DNA bases. As illustrated in Figure S5, the oxidation peak potential positively shifts with the increase of scan rate for each DNA bases. The linear regression equations are: ( ) = 0.0693 log ( / ) + 0.550( = 0.9989) ( ) = 0.1020 log (

/ ) + 0.740( = 0.9988)

( ) = 0.0494 log (

/ ) + 1.035( = 0.9993)

( ) = 0.0219 log (

/ ) + 1.276( = 0.9988)

The intercepts of the above equations reflect the oxidation peak potentials, which imply that some polluted species might be formed during the electrochemical reactions, degrading the catalytic activity of CuNRGO. Effect of adsorption time on the oxidation current of G, A, T, and C. As mentioned above, the electrochemical oxidation reaction of DNA bases occurs via their adsorbed states. Thus, the adsorption potential and time will affect the amount of DNA bases accumulated on the catalyst surface and thus their oxidation currents. It has been reported that G, A, T and C can adsorb on the electrode surface at open circuit condition.32 We studied the influence of accumulation time at 0.4 V on the oxidation currents of the four DNA bases, and the DPVs for different adsorption time is presented in Figure S6. It is clear that the oxidation peak currents of the DNA bases accumulated on CuNRGO/GCE increase with adsorption time (in Figure 4), and they level off at certain adsorption time demonstrating that a saturation adsorption state is reached. Thus, the adsorption times of 20 min, 40 min, 20 min and 10 min for G, A, T and C are used for further experiments. Influence of pH on the oxidation current of G, A, T, and C. Solution pH value will affect the charges carried by the dissolved DNA bases and the catalyst, which will certainly affect the adsorption rates of DNA bases on the catalyst surface and

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Thus, the oxidation peak potentials (Ep) of the four DNA bases show solution pH dependence. The plots of oxidation peak potentials versus solution pH give linear relationship for all the four bases with slope of 56.19, 55.29, 63.57 and 63.67 mV pH−1 for G, A, T, and C bases (Figure S7, insets), which are close to the Nernst value of 0.059 V/pH for a two electrons/two protons reaction as illustrated in Figure S9.33-36

Figure 4. Influence of accumulation time on oxidation peak currents for (a) G (5 μg mL-1), (b) A (5 μg mL-1), (c) T (100 μg mL-1), and (d) C (100 μg mL-1) in PBS (pH = 7.4) at CuNRGO/GCE.

Individual electrochemical catalytic oxidation of G, A, T, and C. Under the optimal experimental conditions, DPVs of CuNRGO/GCE in 0.1 M PBS (pH = 7.4) containing different concentrations of G, A, T, or C were recorded. The results in Figure 6 show that the oxidation currents increase with increasing the concentrations of the four bases. The calibration curves of the oxidation peak currents versus concentrations of individual DNA bases show linear relationships as shown in Figure S9. The regression equations for G, A, T, and C determination are: ( ) = 7.305 + 0.0653( = 0.9994) (

) = 3.506 + 0.1290( = 0.9997)

(

) = 0.03292 − 0.4319( = 0.9998)

(

) = 0.05673 − 1.2855( = 0.9994)

respectively. The linear concentration ranges are 0.02-1.0 μg mL-1 for G, 0.05-0.7 μg mL-1 for A, 25-700 μg mL-1 for T, and 30-175 μg mL-1 for C. The detection sensitivity is 7.305 μA ml μg-1, 3.506 μA ml μg-1, 0.03292 μA ml μg-1 and 0.05673μA ml μg-1, and the detection limit at the signal to noise of 3 is 0.008 μg mL-1, 0.02 μg mL-1, 9 μg mL-1 and 10 μg mL-1 for G, A, T, and C, respectively.

Figure 5. Influence of solution pH on the anodic peak currents of G, A, T, and C on CuNRGO/GCE in 0.1 M PBS containing (a) G (5 μg mL-1), (b) A (5 μg mL-1), (c) T (100 μg mL-1) and (d) C (100 μg mL-1). The data were adopted from Figure S7. thus the oxidation currents. The solution pH dependent DPVs of the four bases on CuNRGO/GCE are shown in Figure S7. As shown in Figure 5, it is clear that the oxidation peak currents of the bases are significantly affected by the solution pH (Figure 5) with the optimized adsorption time. For G base, its oxidation peak current shows relatively stable in the pH range from 3 to 7 and decreases rapidly as the pH becomes larger (Figure 5a). For A base, a bell-shaped relationship between the peak current versus solution pH appears (Figure 5b). The maximum peak current is reached at solution pH close to 6. The oxidation peak current for T base increases with the solution pH in the range of 3-8 and then it reaches a plateau at higher pH (Figure 5c). While the oxidation peak current for C base decreases monotonically with solution pH and reaches the minimum value at solution pH = 9 (Figure 5d). The oxidation currents for the four DNA bases at physiological pH of 7.4 could be acceptable for sensitive detection and thus this pH will be used in the following experiments. It has been reported that the electrochemical oxidation of the DNA bases occurs via electron transfer coupled proton transfer.

Simultaneous determination of DNA bases at CuNRGO/GCE. As Figure 3 shows, the oxidation peak potentials for G, A, T, and C are markedly separated, which allows the simultaneous detection of the bases. As shown in Figure 7(ac), simultaneous determination of the four DNA bases can be achieved on CuNRGO/GCE when they are coexisted in the same solution. The electrochemical responses show linear relationship with the concentrations of G, A, T, and C in the range of 1.2-3.8 μg mL-1, 2.4-6.6 μg mL-1, 40-53 μg mL-1, and 42-53 μg mL-1, respectively. The corresponding regression equations for G, A, T and C are: ( ) = 0.536 − 0.036( = 0.9880) (

) = 0.224 − 0.280( = 0.9920)

(

) = 0.148 − 5.767( = 0.9913)

(

) = 0.120 − 4.984( = 0.9887)

It should be noted that the detection sensitivity and linear ranges for the four DNA bases show some difference as compared to the ones for individual determination, which could be ascribed to the competitive adsorption of the four DNA bases for active sites on the catalyst. The linear detection ranges for the four DNA bases on the CuNRGO/GCE are listed in Table 1, which are comparable or better than the ones reported at other modified electrodes. Interference. The effect of the possible interfering species on the detection of DNA bases were investigated by adding foreign species37 into 0.1 M PBS containing 20 μg mL-1 G and A, 40 μg mL-1 T and C. The experimental results illustrated that some interferences existing in biological samples such as ascorbic acid, l-glycine, l-cysteine and lactic acid in a 20-fold excess, glucose in a 50-fold excess had no significant interference (signal change