Facile synthesis of 3D porous nitrogen-doped graphene as an efficient electrocatalyst for adenine sensing

Abstract

In this work, a simple, low-cost and eco-friendly strategy for fabricating the three-dimensional porous nitrogen-doped graphene (3D-N-GN) is demonstrated by combining the hydrothermal assembly and freeze-drying process without using any framework support. The desired features for 3D-N-GN, such as rich macroporosity, nitrogen-doping structure and high active surface area have been confirmed by scanning electron microscopy, X-ray photoelectron spectroscopy and electrochemical techniques, respectively. In comparison with two-dimensional graphene (2D-GN) and nitrogen-doped graphene (2D-N-GN), 3D-N-GN makes a more negative shift in the oxidation peak potential of adenine together with a remarkable increase in the oxidation peak current, highlighting the importance of the nitrogen-doping and 3D construction of the graphene-based support for improving the electrocatalytic performance. It also indicates that 3D-N-GN can be used as an efficient electrocatalyst for adenine sensing. Furthermore, the sensing conditions are optimized and the resulting sensor displays excellent analytical performance in the detection of adenine at low concentrations ranging from 0.02 to 1.20 μM, with a detection limit of 8 nM. Finally, this proposed method not only exhibits preferable reproducibility, stability and adequate sensitivity, but also demonstrates good efficiency in the detection of adenine in biological fluids.

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1. Introduction

Adenine, as an important purine base which exists in DNA, plays a crucial role in the storage of genetic information and protein biosynthesis, and has a widespread effect on coronary and cerebral circulation, energy transduction, enzymatic reactions as cofactors, and even in cell signaling.¹ The accurate determination of adenine in DNA is extremely significant, because an abnormal change of this purine base is related to a deficiency and mutation of the immune system, which may cause various diseases, such as carcinoma, AIDS, epilepsy and Parkinson's disease. In view of this fact, some classical methods including fluorescence,¹ high-performance liquid chromatography,² liquid chromatography-tandem mass spectrometry,³ surface-enhanced Raman spectroscopy⁴ and electrochemical technique,⁵ have been developed for adenine detection. Among these methods, electrochemical technique has been considered as a perfect alternative due to its superior advantages of simple operation, fast response, low cost, sufficient sensitivity and possibility to miniaturization allowing on-line field monitoring of desired analytes.^6–8 Moreover, DNA and its components adsorbed or covalently attached at the surface of electrode can give important information about the stage of DNA molecule.⁵ Therefore, the development of new sensitive sensors is not only significant for the trace determination of DNA bases in the field of clinical diagnosis, but also able to detect very small changes in the electrochemical properties of DNA bases for insight into fundamental mechanisms of genetic information. However, direct electrochemical detection is not suitable for adenine since its oxidation potential is relatively high at bare electrodes and high oxidation potential is often interfered by oxygen evolution, which results in the low electrochemical recognition. To overcome this obstacle, a variety of nanomaterials with versatile properties have been fabricated and employed as electrode modifiers to magnify the response signals.

To date, a survey in the literature indicates that lots of composites or hybrids including graphene/platinum nanoparticles,⁹ carbon nanotube/ionic liquid,¹⁰ porous silicon supported Pt–Pd nanoalloy,¹¹ TiO₂ nanoparticles/magnesium(II) doped natrolite zeolite,¹² carbon-supported NiCoO₂ nanoparticles¹³ and polyaniline/MnO₂,¹⁴ have been developed as improved electrocatalysts for adenine sensing. Although these materials are effective, the facile synthesis of more novel electrocatalysts based on metal-free or metal oxide-free composites is attractive,¹⁵ and further attempts should be made to efficiently enhance the conductivity, catalytic activity and stability of the designed electrochemical sensors. Among the materials mentioned above, graphene (GN) is a promising matrix for the fabrication of multifarious sensors due to its two-dimensional (2D) sp²-hybridized carbon sheet with large surface, excellent conductivity, high flexibility and other exceptional properties. Unfortunately, the unavoidable aggregation for GN is spontaneously induced by intensive π–π interaction and strong inter-sheet van der Waals interaction, which dramatically decreases the surface area, leading to the increase of the diffusion resistance of reactants/electrolytes and the reduction of the number of exposed active sites.^16–18 Therefore, a careful molecular design of GN is needed to enable its use as a high performance electrocatalyst for sensing applications.

More recently, doping of GN with non-metallic elements has received much attention because of the possibility of changing its physicochemical properties which opens the avenue for potential applications.¹⁹ Previous efforts^20–23 have revealed that incorporating nitrogen into GN can alter the charge density of nitrogen-doped graphene (N-GN) and induce n-type electrical conductivity, resulting in the improvement of the electron-donor property and enhancement of the catalytic activity of GN-based materials. Especially, the preferable catalytic performances of N-GN have been proven in the electrochemical sensing for different kinds of compounds such as pharmaceutical molecules,^21,24 food contaminants,²² enzyme cofactors²³ and small biomolecules with physiological functions.^20,25 Up to now, several nitrogen doping methods have been developed to prepare N-GN, including thermal exfoliation,²⁰ chemical vapor deposition,²⁵ plasma treatment²⁶ and direct-current arc discharge.²⁷ Nevertheless, the applicability of these techniques is considerably limited due to the disadvantages of high cost, inability to scale up, and the need of sophisticated instruments with experienced operators. In addition, most of these methods usually require protective atmosphere and high temperature, which may cause the doped nitrogen to escape easily as time prolongs. Therefore, designing simple and cheap strategies for effective mass production of N-GN sheets in common laboratories still remains a challenge. Furthermore, as compared with 2D-GN, three-dimensional (3D) nanostructured GN provides higher specific surface area, larger pore volume and a multiple lattice-layered graphitic structure. Porous 3D structure with a highly conductive network consisted of defect free GN sheets facilitates the mass transfer on the electrode surface during electrochemical reactions, and also serves as electrochemically active matrix to exhibit superior electrochemically catalytic performances. It can be expected that such unique features not only provide a large amount of active sites for adenine sensing, but also are favorable for the fast transport of oxidation products of adenine. Inspired by these insights, the integration of nitrogen-doping and 3D structure may be an effective way to enhance the detection sensitivity of the designed sensor for adenine.

In this study, we present a combined hydrothermal assembly and freeze-drying approach to prepare a stable 3D-N-GN hybrid without using any framework support. The synthesized hybrid was carefully characterized by various means and the electrocatalytic properties of 3D-N-GN toward adenine were evaluated using voltammetric techniques. The results show that the 3D-N-GN hybrid can provide a large electrochemically active surface area for the catalysis of adenine and effectively accelerate the electron transfer between the electrode and solution, which could allow a more rapid and sensitive current response. It has indicated that 3D-N-GN can be used as a new and efficient catalyst for adenine sensing. Besides, the dynamics mechanisms of adenine oxidation at the prepared sensor were investigated in detailed. Finally, the proposed method was successfully applied for the assessment of adenine content in spiked rabbit serum and human urine samples, and satisfactory recoveries between 94.0–105.0% were obtained.

2. Experimental

2.1. Reagents

Adenine (analytical grade) was purchased from Aladdin Chemical Reagent Co. (China). Graphite powders (spectrum pure), KMnO₄ (analytical grade), H₂SO₄ (98 wt%) and H₂O₂ (30 wt%) were obtained from Sinopharm Chemical Reagent Co. (China). K₄[Fe(CN)₆], K₃[Fe(CN)₆], KCl, NaOH, Na₂HPO₄ and NaH₂PO₄ were of analytical grade and purchased from Shanghai Chemical Reagent Co., Ltd. (China). 0.1 M of phosphate buffer solution (PBS) was prepared by mixing 0.1 M of Na₂HPO₄ aqueous solution with 0.1 M of NaH₂PO₄ aqueous solution, which was adjusted to a required pH value before used. The deionized water with resistivity less than 18 MΩ cm (Millipore Milli-Q system) was used for preparations of all solutions and the experiments were carried out at room temperature.

2.2. Instruments

The surface morphologies of the synthesized products were investigated by an EVO-MA10 scanning electron microscope (SEM, ZEISS, Germany) operating at 10 kV. High-resolution transmission electron microscopy (HRTEM) images were recorded on a JEM-2100 microscope (JEOL, Japan) operating at 200 kV. X-ray photoelectron spectroscopy (XPS) analysis was carried out with a K-Alpha 1063 diffractometer (Thermo Fisher Scientific Inc., UK) equipped with a monochromatic Al Ka source operated at 72 W. The Brunauer–Emmett–Teller (BET) surface areas were obtained from N₂ sorption isotherms at 77 K using an autosorb iQ₂ automated gas sorption analyzer (Quantachrome Instruments, UAS). Raman spectra were obtained on a LabRAM HR800 spectrometer (Horiba Jobin Yvon, France) from 100 to 3000 cm⁻¹. Electrochemical measurements were performed on a CHI 660D electrochemical workstation (ChenHua Instruments Co., Shanghai, China). A conventional three electrode configuration was employed, consisting of a bare or modified glassy carbon electrode (GCE, 3 mm in diameter) as the working electrode, an Ag/AgCl saturated KCl electrode as the reference electrode, and a platinum wire as the auxiliary electrode. All the pH values were measured with a PHS-3C precision pH meter (Shanghai Leici Instrument Factory, China), which was calibrated with standard buffer solution every day.

2.3. Synthesis of 3D-N-GN

Graphite oxide (GO) was synthesized from natural graphite powders by a modified Hummers' method, of which details were described elsewhere.^28,29 Then the 3D-N-GN was prepared by a combined hydrothermal assembly and freeze-drying process. In a typical procedure, 50 mL of GO (1.0 mg mL⁻¹) aqueous dispersion was first treated by sonication for 20 min and then was adjusted to pH 8.0 using dilute ammonia solution. After that, urea (2.0 g) was added under vigorous stirring into the GO solution and then the mixture was transferred into a Teflon-lined stainless-steel autoclave and kept at 150 °C for 10 h. After the solvothermal procedure, the autoclave cooled naturally to room temperature. The obtained products were dialyzed with deionized water for one day at least. Finally, the black integrated 3D-N-GN was obtained via a freeze-drying process to maintain the 3D monolithic architecture while 2D-N-GN was obtained via a bake drying process at 60 °C. The 2D-GN was obtained by the similar procedure without using urea and freeze-drying process.

2.4. Fabrications of the modified electrodes

Prior to use, the bare GCE was polished on chamois leather with 0.05 μm α-alumina powders and then ultra-sonicated successively in 1 : 1 aqueous HNO₃ (v/v), absolute alcohol and deionized water to get a mirror-shiny surface. Then 5 μL of 3D-N-GN suspension (1 mg mL⁻¹), which was prepared by dispersing 3D-N-GN powders in DMF with the aid of ultrasonic agitation for 0.5 h, was coated on the electrode surface and dried under infrared lamp to obtain 3D-N-GN/GCE. The 2D-N-GN/GCE and 2D-GN/GCE were fabricated in the same way.

3. Results and discussion

3.1. Morphologies and structure characteristics of 3D-N-GN

The detailed surface morphologies and structure characteristics of the synthesized graphene-based products were characterized by SEM at different magnifications and the typical results are shown in Fig. 1. From Fig. 1A and B, it can be clearly seen that the thin, wrinkled and folded paper-like textures of nanosheets are typically observed for 2D-GN. In Fig. 1C and D, 2D-N-GN is observed to have a flake-like structure in irregular size. It can be aware from the images that 2D-N-GN has larger surface area in comparison with 2D-GN, which makes it easy for the electron transfer. The SEM images of 3D-N-GN with low and high magnifications in Fig. 1E and F confirm the wrinkled nanosheets with well-defined porous structures. In light of the latest publication,³⁰ the formation of cross-linked network structures for 3D-N-GN derives from the reduction of restored conjugated structure of graphene oxide sheets due to their functionalization by urea in an aqueous medium with ammonia, which induces partial over-lap or aggregation of flexible graphene sheets via π–π stacking interactions, resulting in formation of the strong cross-links of the 3D porous network. Moreover, the high magnification SEM image in Fig. S1† shows that 3D-N-GN has a highly porous structure with pore sizes ranging from 3.2 to 8.5 μm, indicating that the prepared 3D nanostructured hybrid contains rich macropores. In Fig. 2A and B, the BET analysis shows a specific surface area of 443.7 m² g⁻¹ for the 3D-N-GN, which is much higher than that of the 2D-GN with an aggregated structure (87.2 m² g⁻¹). Such system is an attractive target, as it would allow the utilization of the unique features of the 3D hybrid, such as rich macroporosity and highly accessible surface area, which are beneficial to the analyte adsorption and electron transfer at the modified electrode surface. To show further insight into the detailed nanostructures of the 3D-N-GN, HRTEM image is provided in Fig. 2C. It can be found that the 3D network is mainly composed of predominantly 5–8 layers of crystalline nano-slices. The spacing of the adjacent lattice planes for the layers is about 0.34 nm, corresponding to (002) planes of graphite.

Fig. 1 SEM images of (A, B) 2D-GN, (C, D) 2D-N-GN and (E, F) 3D-N-GN films at different magnifications.

Fig. 2 N₂ adsorption–desorption isotherms of 2D-GN (A) and 3D-N-GN (B), HRTEM images of 3D-N-GN at different amplifications (C) and Raman spectrum of 3D-N-GN (D).

To confirm the successful doping of GN with nitrogen, the surface composition of 3D-N-GN and chemical state of nitrogen atoms were examined by XPS and Raman spectroscopy. The Raman spectrum of 3D-N-GN (Fig. 2D) displays the D-band and G-band centered at 1332 and 1582 cm⁻¹, respectively. The intensity ratio of D to G band is about 1.16, indicating the existence of substantial defects or disordered sites in the structure of 3D-N-GN, presumably stemming from the presence of N dopants and concomitant absence of C atoms in the hybrid structure.³¹ Fig. 3A shows three obvious XPS signals at 532.5, 400.1 and 285.6 eV, which can be assigned to O 1s, N 1s and C 1s, respectively. This result further corroborates that nitrogen atoms have been successfully incorporated to the GN sheets. Moreover, 4.9 wt% nitrogen was estimated to be present in the 3D-N-GN hybrid on the basis of the XPS analysis. The addition of N element can change the electron density of the N-GN surface, making the electron transfer of N-GN more easily and resulting in the great improvement for sensitivity of the prepared sensors. The high-resolution N 1s scan in Fig. 3C shows a wonderful peak at ∼400.1 eV which is attributed to the presence of pyrrolic N. Notably, the related study²⁰ has confirmed that pyrrolic N possesses the better catalytic activity than the other N structures (pyridinic N and graphitic N), illustrating that the prepared hybrid will own more active sites for electrochemical sensing. As shown in Fig. 3D, the peaks observed at 284.6 and 286.5 eV indicate the presence of C–C (sp²) and C–O, and the peaks at 285.6 and 287.1 eV are assigned to the sp² and sp³ bonded C–N,³² respectively, originating from the substitutional doping of nitrogen atoms.

Fig. 3 XPS spectra of (A) wide scan, (B) O 1s, (C) N 1s and (D) C 1s of 3D-N-GN.

3.2. Electrochemical characterization of the modified electrodes

Electrochemical impedance spectroscopy (EIS), as a valid method to monitor the interface features of the modified electrode, generally includes a semicircular portion and a linear portion. The semicircle portion corresponds to the limited process of electron transfer at a higher frequency, while the diameter of semicircle in EIS equals to the electron transfer resistance (R_et) at the electrode surface. Herein, EIS was performed at 0.17 V with the AC voltage amplitude of 0.005 V and frequency range of 10⁵to 0.1 Hz using [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ (1 : 1) as the electrochemical probe. Fig. 4A gives a clear illustration to the impedance changes for GCE (a) and GCEs modified by 2D-GN (b), 2D-N-GN (c) and 3D-N-GN (d). As can be seen, R_et of 2D-GN (850 Ω) is reduced compared with that of bare GCE (1100 Ω), indicating that 2D-GN is an appropriate matrix to accelerate the electron transfer rate. Interestingly, the R_et of 2D-N-GN further decreases to 530 Ω, which is caused by the nitrogen doping into the GN structure. It is noteworthy that 3D-N-GN/GCE exhibits the smallest semicircle (210 Ω), which might be ascribed to the 3D and porous structure, facilitating the electron transfer and improving diffusion passageways of the probe ions. The EIS results also verify that 3D-N-GN has been stably coated on GCE surface.

Fig. 4 (A) Nyquist diagrams and (B) CV curves of 0.5 mM [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ (1 : 1) in 0.1 M KCl solution recorded at (a) GCE, (b) 2D-GN/GCE, (c) 2D-N-GN/GCE and (d) 3D-N-GN/GCE.

Cyclic voltammetry (CV) is a useful tool to investigate the electrochemical natures of modified electrodes. The CV curves of bare GCE (a), 2D-GN/GCE (b), 2D-N-GN/GCE (c) and 3D-N-GN/GCE (d) obtained at a scan rate of 0.1 V s⁻¹ and the potential scan range from −0.3 V to 0.6 V are shown in Fig. 4B. A pair of peaks corresponding to the redox reaction of ferricyanide can be observed at all electrodes. As expected, 3D-N-GN/GCE exhibits the highest peak current, suggesting that the best conductivity is achieved by the 3D-N-GN hybrid, which agrees well with the conclusion from EIS. This is due to the fact that the 3D nanostructure with a short path for ion diffusion and large surface area provides more efficient contact between the probe ions and the active materials, which is also conductive to the rapid heterogeneous electron transfer. Furthermore, the effective areas of the modified electrodes can be obtained by CV using 0.5 mmol L⁻¹ K₃Fe(CN)₆ as the probe at different scan rates. For a reversible process, the following equation exists:³³

I_pa = (2.69 × 10⁵)n^3/2ACD^1/2ν^1/2 (1)

where I_pa (A) refers to the anodic peak current, n is the total number of transferred electron (for K₃Fe(CN)₆, n = 1), A (cm²) is the electroactive area of the electrode, D (cm² s⁻¹) is the diffusion coefficient (for K₃Fe(CN)₆, D = 7.6 × 10⁻⁶ cm² s⁻¹), C (mol cm⁻³) is the concentration of K₃Fe(CN)₆ and ν (V s⁻¹) is the scan rate. Therefore, the effective area can be calculated from the slope of the I_pa–ν^1/2 relation. The effective areas of 2D-GN/GCE, 2D-N-GN/GCE and 3D-N-GN/GCE were evaluated to be 0.181, 0.266 and 0.315 cm² respectively, which are about 1.5, 2.7 and 3.4 times greater than the geometric area of unmodified GCE (0.071 cm²), indicating that the introduction of the 3D-N-GN hybrid will provide more conductive pathways for the electron transfer at GCE surface. The CV and EIS results have further testified the inferences from SEM and XPS.

3.3. Electrochemical behaviors of adenine on the prepared sensors

In order to compare the electrocatalytic performances of all the prepared sensors, CV was employed to study the electrochemical behaviors of 1.0 μM adenine at bare GCE (a, Fig. 5A), 2D-GN/GCE (b, Fig. 5A), 2D-N-GN/GCE (c, Fig. 5A) and 3D-N-GN/GCE (d, Fig. 5A) in PBS solution of pH 3.0 at a scan rate of 0.1 V s⁻¹. At bare GCE, a broad oxidation peak with a weak peak current for the adenine is observed. When a layer of 2D-GN is coated on the GCE surface, a relatively obvious oxidation peak appears at 1.213 V, which is mainly due to the increase of electrode surface area. Furthermore, the anodic peak potentials for adenine oxidation at 2D-N-GN/GCE and 3D-N-GN/GCE are 1.202 and 1.174 V, indicating that the peak potentials for adenine oxidation at these two electrodes shift by ∼11 and 39 mV toward negative values compared with that of 2D-GN/GCE, respectively. Also, it can be seen that 3D-N-GN/GCE shows a much higher anodic peak current for the oxidation of adenine compared with 2D-GN/GCE and 2D-N-GN/GCE, illustrating that the combination of nitrogen-doping and 3D porous structure has significantly improved the performance of the electrode toward adenine oxidation. The CV curves of 3D-N-GN/GCE in the PBS buffer of pH 3.0 in absence (a) and presence (b) of 1.0 μM adenine are given in Fig. 5B. There is no distinct redox peaks appearing in the blank solution, indicating that 3D-N-GN is non-electroactive in the scanned potential window. After the addition of adenine, it is very evident that a well-defined and sensitive oxidation peak for adenine appearing at 3D-N-GN/GCE during the first anodic sweep and the following reverse scan shows no corresponding reduction peak, indicating that the adenine oxidation is an irreversible process under this condition, in accordance with the outcomes of the reported papers dealing with poly(xanthurenic acid)–MoS₂,³⁴ ionic liquids coated nanocrystalline zeolite materials,³⁵ molybdenum disulfide nanosheets,³⁶ graphene quantum dots/silver nanoparticles,³⁷ and so on. To obtain information about the rate-determining step, the Tafel plot for 3D-N-GN/GCE in 0.1 M of PBS (pH = 3.0) containing 1.0 μM adenine was derived from points in the Tafel region of CV at a scan rate of 5 mV s⁻¹. The slope of the Tafel plot is equal to n(1 − α)F/2.3RT. Therefore, the value of electron transfer coefficient α is 0.47.

Fig. 5 (A) CV curves of (a) GCE, (b) 2D-GN/GCE, (c) 2D-N-GN/GCE and (d) 3D-N-GN/GCE in 0.1 M PBS (pH = 3.0) containing 1.0 μM adenine at the scan rate of 0.1 V s⁻¹; (B) CV curves of 3D-N-GN/GCE in (a) absence and (b) presence of 1.0 μM adenine.

3.4. Effect of pH value

The effect of buffer pH on the electrochemical response of 1.0 μM adenine at 3D-N-GN/GCE was investigated at different pH solutions ranging from 2.0 to 7.0 (Fig. 6A). Fig. 6B and C show the relationships of peak potential (E_pa) and oxidation peak current (I_pa) vs. pH values, respectively. The oxidation peak potential for adenine moves to the negative direction with the increase of buffer pH, indicating the involvement of protons in the electrochemical reaction. The plot of E_p vs. pH shows a straight line, which can be expressed by the following equation: E_pa (V) = 1.3054 − 0.0564 pH (R² = 0.9751). The slope is found to be −56.4 mV pH⁻¹, which is very close to the theoretical value of −59 mV pH⁻¹, indicating that the number of protons participating in the electrode reaction is equal to the number of transferred electrons. The similar results were observed at electrodes modified by electrochemically reduced carboxyl graphene,³⁸ N-methyl-2-pyrrolidone-exfoliated graphene nanosheets,³⁹ silver nanoparticles-β-cyclodextrin-graphene⁴⁰ and poly-melamine film.⁴¹ It is also found that the peak current of adenine at 3D-N-GN/GCE is augmented along with the increase of pH from 2.0 to 3.0 and then starts to decrease for pH values higher than 3.0. Especially in pH 7.0, the electrochemical response of 3D-N-GN/GCE to adenine is very small. Hence, pH 3.0 was chosen as the optimum pH value for further measurements.

Fig. 6 (A) CV curves of 1.0 μM adenine recorded at 3D-N-GN/GCE with different pH values (pH values from curve a to h are 2.0, 2.5, 3.0, 3.3, 4.0, 5.0, 6.0 and 7.0, respectively); (B) the relationship between the peak potential and solution pH; (C) the relationship between the peak current and solution pH; scan rate: 0.1 V s⁻¹.

3.5. Effect of scan rate and reaction mechanism of adenine

To understand the reaction pathway and conjecture the reaction mechanism of adenine at 3D-N-GN/GCE, it is necessary to investigate the influence of scan rate on the electrochemical oxidation of adenine using CV method (Fig. 7A). As illustrated in Fig. 7A, both the oxidation peak currents and potentials increase with the scan rate elevating. Plotting the peak current (I_pa) vs. the scan rate (ν) yields a well-defined linear relationship (Fig. 7B), and the regression equation can be expressed as I_pa (μA) = 15.1626ν (V s⁻¹) + 2.0514 (R² = 0.9937). This result indicates that the oxidation of adenine at 3D-N-GN film occurs as an adsorption-controlled process, which is consistent with the phenomena observed at the composite films of CuO shuttle-like nanocrystals/poly(neutral red),⁴² (2,6-pyridinedicarboxylic acid)/graphene⁴³ and graphene/ionic liquid/chitosan.⁴⁴ The peak current is related to the surface concentration of electroactive species, which mainly depends on the adsorption capacity of the modified electrode. As we known, the surface coverage (τ) is an important parameter to assess the adsorption capacity. From the slope of anodic peak current vs. scan rate, surface coverage τ can be acquired using the following equation:where I_p is the peak current, A (0.315 cm²) is the electroactive surface area, ν is scan rate, n (assuming n to be 2) is the number of transferred electrons, and R, T and F have their habitual meanings. By considering the above values τ was calculated as 1.28 × 10⁻⁵ mol cm⁻², which is much larger than the previously reported values at the surfaces of TiO₂ nanoparticles/magnesium(II) doped natrolite zeolite (1.04 × 10⁻⁷ mol cm⁻²),¹² nickel loaded porous carbon nanofibers (1.976 × 10⁻⁷ mol cm⁻²)⁴⁵ and NiFe₂O₄ decorated MWCNTs (3.15 × 10⁻⁹ mol cm⁻²).⁴⁶ The obtained result is also larger than the coverage of monomolecular layer, indicating that the multiple layers of adenine were coated on the electrode surface due to the excellent adsorption property of 3D-N-GN.

Fig. 7 (A) CV curves of 1.0 μM adenine recorded at 3D-N-GN/GCE with different scan rates (scan rates from curve a to n are 0.02, 0.04, 0.06, 0.08, 0.10, 0.15, 0.20, 0.25, 0.30, 0.40, 0.50, 0.60, 0.70 and 0.80 V s⁻¹, respectively); (B) the plot of anodic peak current vs. scan rate; (C) the plot of anodic peak potential vs. Napierian logarithm of scan rate.

As displayed in Fig. 7C, the oxidation peak potentials (E_pa) are linear as a function of the Napierian logarithm of scan rates (ln ν) in the range from 0.02 to 0.80 V s⁻¹ and the linear equation can be plotted as E_pa (V) = 0.0246 ln ν (V s⁻¹) + 1.2319 (R² = 0.9899). For an irreversible electrode process, E_pa is defined by the following equation according to Laviron's model:⁴⁷

where E⁰ is the formal standard potential, α is the transfer coefficient, k_s is the standard heterogeneous rate constant of the reaction, n is the number of transferred electrons and ν is the scan rate. Other symbols have their common meanings. Thus, the value of αn can be easily calculated from the slope of E_pa vs. log ν. In this system, the slope was found to be 0.0246. Taking T = 298 K and substituting the values of R and F, (1 − α)n was calculated to be 1.044. In the Section 3.3., α has been evaluated to be 0.47, so n was calculated to be 2.0. In view of equal numbers of electrons and protons involved in the adenine oxidation, it can be deduced that there is a two-proton and two-electron process for the adenine electro-oxidation at 3D-N-GN/GCE, which is in common with the consequences achieved at Fe₃O₄/MWCNT/GCE,⁴⁸ overoxidized polypyrrole/graphene/GCE,⁴⁹ poly(eriochrome black T)/GCE⁵⁰ and ionic liquids coated nanocrystalline zeolite materials.³⁵ According to relevant reports,^35,49–51 the oxidation of adenine is followed by the two-step mechanism including the overall loss of four electrons with first two electrons' oxidation considered as a rate-determining step, leading to the creation of 2-oxoadenine. With this in mind, the possible reaction mechanism for the adenine electro-oxidation in our work is proposed and listed in Fig. S2.† Moreover, the formal standard potential (E⁰ = 1.158 V) was calculated from another linear relation of E_pa–ν by extrapolating ν = 0. Then, the heterogeneous electron transfer rate constant k_s could be calculated to be 813 s⁻¹ based on the eqn (3). The value of k_s is larger than a previously reported value of 290.3 s⁻¹,⁵² indicating a fast electron transfer rate at 3D-N-GN/GCE.

3.6. Effect of the accumulation conditions

In consideration of the fact of adenine adsorbed on the 3D-N-GN surface, it is significant to optimize accumulation conditions for achieving the high detection sensitivity. Both accumulation potential and accumulation time could affect the amount of adsorption of adenine at the electrode surface. Bearing this in mind, the effect of accumulation potential and time on the peak current response has been studied. When the accumulation potential was varied from −0.50 to +0.50 V and also fixed at open circuit condition, the peak current changed a little. Hence, the accumulation at open circuit was approved. Also the influence of accumulation time ranging from 0 to 400 s on the oxidation of adenine at 3D-N-GN/GCE was investigated. The peak current increased gradually as the accumulation time prolonged from 0 to 200 s. However, with further increasing the accumulation time beyond 200 s, the peak current tended to be almost stable. Therefore, the optimal accumulation time of 200 s was employed in further investigations.

3.7. Effect of the modifier amount

The amount of modifier can change the properties and functions of the electrode surface. Consequently, the effect of the amount of 3D-N-GN as a modifier was studied by adding 1–10 μL of dispersed 3D-N-GN suspension onto the GCE surface. It was affirmed that the oxidation peak current increased with increasing the amount of 3D-N-GN up to 5 μL and then the saturation in the anodic peak current happened. Therefore, 5 μL of 3D-N-GN suspension was selected as the optimum amount for the preparation of the modified GCE.

3.8. Calibration curve for determination of adenine

As is known to all, differential pulse voltammetry (DPV) has much higher sensitivity and discrimination than CV, so the calibration curve for adenine determination was investigated by DPV under the optimal conditions. Fig. 8A shows DPV curves for the different concentrations of adenine at 3D-N-GN/GCE in PBS of pH 3.0. Obviously, there is an enhancement in its oxidation peak current with the increase of the adenine concentration, suggesting that 3D-N-GN/GCE can be applied for the quantitative determination of adenine. Fig. 8B shows that the oxidation peak currents have a good linear relationship with the adenine concentrations in the range from 0.02 to 1.20 μM. The regression equation can be given as I_pa (μA) = 6.8954C (μM) + 0.1320 (R² = 0.9983). The detection limit was estimated to be 8 nM at the signal-to-noise ratio of 3. To make clear the analysis performance of the developed sensor, the comparison of different sensors reported for adenine detection is summarized in Table 1. It can be seen that the linear range of 3D-N-GN/GCE is comparable to those obtained at N-methyl-2-pyrrolidone-exfoliated graphene nanosheets/GCE⁵⁴ and nickel loaded porous carbon nanofibers/GCE,⁵⁵ affording a much wider scope in the low concentrations in comparison with other sensors. Moreover, it is worth noting that the detection limit and sensitivity of the proposed method outperform most of the reported methods, indicating that the 3D-N-GN hybrid can serve as an excellent platform for the electrochemical detection of adenine.

Fig. 8 (A) DPV curves obtained at 3D-N-GN/GCE in 0.1 M PBS containing different concentrations of adenine (concentrations from curve a to k are 0.00, 0.02, 0.06, 0.12, 0.20, 0.30, 0.45, 0.60, 0.75, 1.00 and 1.20 μM, respectively, and operating conditions for DPV are pulse amplitude of 0.05 mV, pulse width of 0.05 s and pulse period of 0.5 s); (B) the linear relationship between the peak current and concentration of adenine.

Table 1 Performance comparison of different electrochemical sensors for the determination of adenine

Electrode materials	Technique	Linear range (μM)	Detection limit (nM)	Sensitivity (μA μM^−1)	Correlation coefficient	References
a DPV: differential pulse voltammetry.b NMP: N-methyl-2-pyrrolidone.c MWCNTs: multi-walled carbon nanotubes.d LSV: linear sweep voltammetry.
Polyaniline/MnO2	DPV^a	10–150	7800	0.1	0.9943	14
Poly(xanthurenic acid)–MoS2	DPV	0.5–10	32	0.1835	0.9982	34
Electrochemically reduced carboxyl graphene	DPV	2.5–50	100	1.236, 0.405	0.998, 0.997	38
NMP^b-exfoliated graphene nanosheets	DPV	0.05–2.5	10	—	—	39
(2,6-Pyridinedicarboxylic acid)/graphene	DPV	0.1–6.0	20	1.2692	0.998	43
Graphene/ionic liquid/chitosan	DPV	1.5–350	450	0.0322	0.9986	44
Nickel loaded porous carbon nanofibers	DPV	0.05–2	30	1.7343	0.997	45
NiFe2O4 decorated MWCNTs^c	LSV^d	0.1–4.0	10	1.250	0.9907	46
Boron-doped diamond	DPV	0.12–25	19	0.160	0.999	51
Pd-nanowire arrays	DPV	4–200	1000	0.02026	0.9956	53
Xanthurenic acid/graphene	DPV	1–10, 10–300	600	3.0512, 0.2311	0.9906, 0.9927	54
Overoxidized polyimidazole/graphene oxide	DPV	9.6–215	1280	0.232	0.9922	55
Three-dimensional nitrogen-doped graphene	DPV	0.02–1.2	8	6.8954	0.9983	This work

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3.9. Repeatability, reproducibility, stability and interference

The repeatability of 3D-N-GN/GCE was examined by successively measuring the DPV responses of 1.0 μM adenine in 0.1 M of PBS (pH = 3.0) using the same sensor and the results are listed in Table S1.† It was found that the relative standard deviation (RSD) was 2.17% (n = 6), indicating that the good repeatability was achieved at 3D-N-GN/GCE. Additionally, the reproducibility was tested by the determination of 1.0 μM adenine using DPV method with six sensors which were independently fabricated under the same conditions (Table S2†). The oxidation peak currents of adenine were comparable, with RSD of 4.36%, revealing a satisfied reproducibility for the prepared sensor. The long-term stability is also a critical factor to evaluate the performance of the prepared sensor (Table S3†). The DPV responses of 1.0 μM adenine were collected every three days to check the long-term stability, and the electrode was stored at 4 °C when not in use. The sensitivity remained 87% of its original value after a month, suggesting that the fabricated electrode has a considerable stability for the adenine detection.

The selectivity of 3D-N-GN/GCE is one of the most important factors for its practicability. To evaluate the selectivity of the proposed method, the effects of various potentially interfering substances commonly coexisting in biological samples were investigated using DPV for the determination of 1.0 μM adenine under optimum conditions (Table S4†). The tolerance limit was defined as the maximum concentration of the foreign substances that caused an approximately ±5% relative error in the detection. The results demonstrate that 500-fold concentrations of K⁺, Na⁺, Ca²⁺, Cu²⁺, Mg²⁺, Zn²⁺, Fe³⁺, Cl⁻, PO₄³⁻, NO³⁻, SO₄²⁻, CO₃²⁻, glucose, sucrose, arginine, alanine, threonine, serine, valine and cysteine have no influence on the detection of adenine. Ascorbic acid, uric acid, dopamine, tyrosine and tryptophan show oxidation process in the potential range of 0.0–1.0 V, illustrating the oxidation potentials are lower than those of adenine. It has been found that 50-fold concentrations of ascorbic acid, uric acid, tyrosine, tryptophan, guanine, thymine and cytosine do not interfere significantly, while dopamine has an effect on the measurements of adenine to some degree (±8%). From these results, it may be concluded that the proposed electrode is free from interference by most potentially interfering substances in practical samples. The excellent long-term stability, good reproducibility and acceptable selectivity of the developed electrochemical sensor make it an attractive option for practical applications.

3.10. Analytical applications

As described in previous sections, the proposed method was found to be very sensitive and sufficiently selective to allow determination of adenine. In order to evaluate the matrix effect in biological fluids such as rabbit serum and human urine, the standard addition method has been employed. The rabbit serum samples were prepared by centrifuging the rabbit blood at 2000 rpm for 15 min, thereby separating the serum before collecting it with a syringe. The serum samples were then stored at 4 °C before used. The human urine samples were collected by healthy volunteers and used without any pretreatment. Subsequently, 100 μL of rabbit serum and human urine samples were diluted to 10 mL with PBS (pH 3.0) and then analyzed by the DPV technique. The obtained results are listed in Table S5.† It can be clearly found that the recoveries at three spiked levels are between 94.0% and 105.0% with the RSD in the range of 2.5–4.2%, which evidences the developed method is of good accuracy and reliability.

4. Conclusions

This study presents a facile and effective approach to the synthesis of 3D-N-GN hybrid by using a hydrothermal assembly and freeze-drying process. The as-synthesized hybrid exhibits an outstanding electrocatalytic activity toward the oxidation of adenine with a lower overpotential and higher current response compared with 2D-N-GN and 2D-GN. As desired, the high sensitivity, low detection limit, reasonable linear range, good reproducibility and long-term stability have been achieved at the 3D-N-GN modified electrode together with the successful application for the determination of adenine in biological samples with satisfying results. This superiority in analytical performance of the 3D-N-GN hybrid may be ascribed to two main factors. One is the extraordinary electron-transfer properties derived from nitrogen-doping, and the other may be the enrichment of 3D porous structure for adenine on the electrode surface. With these advantages, the 3D-N-GN hybrid can be used as an excellent metal-free electrocatalyst for the electrochemical sensing applications. It can be anticipated that our proposed strategy may provide an efficient approach for the synthesis of a series of 3D heteroatom-doped graphene hybrids for the electrochemical sensing applications.

Acknowledgements

This work was financially supported by National Natural Science Foundation of China (No. 21505035, 21171174, 21472038), Provincial Natural Science Foundation of Hunan (No. 2016JJ3028, 2016JJ4010, 09JJ3024), Scientific Research Project of Education Department of Hunan Province (No. 14C0168, 15A027), Provincial Environmental Science and Technology Foundation of Hunan, the Aid Programs for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province, the Opening Subjects of State Key Laboratory of Powder Metallurgy and Province Key Laboratory of Functional Metal Organic Materials (No. GN14K03), and the Open-end Fund for the Valuable and Precision Instruments of Central South University.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra01864e

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