NiCo-embedded in hierarchically structured N-doped carbon nanoplates for the efficient electrochemical determination of ascorbic acid, dopamine, and uric acid

Abstract

The development of a highly stable and efficient catalyst for sluggish electrooxidation in the electro-determination for ascorbic acid (AA), dopamine (DA) and uric acid (UA) is extremely important for the long-term operation and commercialization of a biosensor device, but it remains a challenge. Herein, we demonstrated an interesting structure of NiCo alloy nanocrystals embedded in hierarchically structured N-doped carbon nanoplates (NiCo-NPs-in-N/C), which is facilely synthesized via a one-step in situ reduction pyrolysis strategy. The two-dimensional N-doped porous carbon shells not only offered the effective confinement effect of NiCo nanocrystals avoiding detachment, dissolution, migration, and aggregation during catalysis process, but also allowed a fast transport pathway for the access of electrolyte to the NiCo surface. As a result, such an intriguing structure shows superior catalytic activity towards the electrooxidation of AA, DA, and UA. The well-separated voltammetric peaks between AA–DA, DA–UA, and AA–UA at the NiCo-NPs-in-N/C are up to 178, 122, and 300 mV, respectively, which is much better than graphene@N-doped carbon core@shell nanoplate (graphene@N/C) and NiCo alloy. Furthermore, the NiCo-NPs-in-N/C also exhibits good reproducibility and stability. The attractive features of NiCo-NPs-in-N/C make it a promising electrocatalyst for the simultaneous determination of AA, DA, and UA.

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1. Introduction

Precise dopamine (DA) determination is of great clinical importance because it is a crucial catecholamine neurotransmitter in the mammalian central nervous system and low levels of it will cause heart disease, Parkinson's¹ and a variety of neurological diseases.² Electrochemical (EC) method was demonstrated to be a popular technology in DA determination due to its fast response, simplicity, low cost, and high sensitivity.^3–5 However, a major problem facing EC detection for DA is the similar electrooxidation peak potentials for ascorbic acid (AA), DA, and uric acid (UA) (coexisting in body fluids), resulting in poor selectivity and reproducibility.⁵ Therefore, the development of robust and durable catalysts for the simultaneous separation of their signal potentials with improved activity and stability is highly desirable.^6–17 Metallic (such as Au, Pt, Ni, Co) nanoparticles (NPs) supported on active substrates, representing an important class of catalysts for different fields, especially for EC determination of AA, DA, and UA, have emerged recently and have attracted intensive investigation.^2,6 Prominent examples include CuO@3D graphene foams,¹⁸ Au@carbon dots-chitosan composite film,¹⁹ and size-selected Pt@graphene/nanocomposites.²⁰ A challenging issue for EC determination is that in corrosive reaction conditions, metallic catalysts tend to show very limited stability and activity owing to their aggregation and the relatively weak interaction between the metallic NPs and the supports. This means that the simple combination of metallic NPs and active substrates still does not satisfy to solve the significant issues of EC determination.

Recent studies revealed that metallic-based NPs (such as Co and Fe) confined inside carbon architectures (such as carbon nanotubes) can exhibit enhanced catalytic activity towards electrochemical reactions (such as oxygen reduction reaction and hydrogen evolution reaction) due to their high resistance to oxidation, improved electron and charge transport, and excellent mechanical strength.^21–23 In this regard, the remarkable characteristics of metallic-embedded carbon nanostructure motivate us to ask whether we can rationally design a new class of metal/C-based nanomaterials to be more robust and practical catalysts towards EC determination of AA, DA, and UA.

On the other hand, many single-metal catalysts hold the drawbacks of unsatisfactory sensitivity, poor selectivity and easy poisoning, which are critical issues for practical applications in biosensors. Recently, Zhang et al. designed an electrochemical biosensor for ascorbic acid based on carbon-supported PdNi nanoparticles and good analytical results were obtained.²⁴ Bimetallic systems can bring interesting physical and chemical properties into effect from the intermetallic combinations of different metals.^25,26 Inspired by the abovementioned research, the bimetallic systematic catalysts are expected to give a high performance for EC determination of AA, DA, and UA. One of the most promising materials based on bimetal is the NiCo alloy, which has been widely used for catalysis,²⁶ microwave absorption,²⁷ and other applications²⁸ with enhanced performance, due to a synergistic effect for catalytic property between the two metals and active oxidation. However, research concerning the NiCo alloy catalysts for EC determination of AA, DA, and UA has rarely reported.

Herein, we demonstrate one-step nanospace pyrolysis strategy (Scheme 1) for the in situ synthesis of NiCo alloy NPs embedded in hierarchical structured nitrogen-doped carbon nanoplates (NiCo-NPs-in-N/C). Uniform NiCo alloy NPs with a well-defined morphology and particle size of 15–20 nm were homogenously distributed within the N-doped carbon nanoplate. By changing the annealing temperature, the formation process of NiCo alloy NPs in the N-doped carbon nanoplates was investigated. Such unique structure shows high electrocatalytic activity toward the electrochemical oxidation of AA, DA, and UA, which benefits from the synergistic effect of NiCo alloy NPs, N-doped functionalities, and the hierarchical structures. As a consequence, well-separated voltammetric peaks between AA–DA, DA–UA and AA–UA at the NiCo-NPs-in-N/C are up to 178, 122, and 300 mV, respectively. For simultaneous sensing of three analytes, the linear response ranges for AA, DA and UA are 500–1500, 0.5–900 and 10–300 μM, respectively, and the detection limits (S/N = 3) are 0.091, 0.080 and 0.014 μM, respectively. Furthermore, NiCo-NPs-in-N/C also exhibits good reproducibility and stability.

Scheme 1 Schematic of the synthetic protocol of NiCo-NPs-in-N/C.

2. Experimental

2.1. Materials

Dopamine hydrochloride (DA, Alladin), uric acid (UA, Sigma), L-ascorbic acid (AA, Tianjin chemical reagent co., Ltd), N,N-dimethylformamide (DMF, Guangdong guanghua science and technology co., Ltd), nickel(II) nitrate hexahydrate (Ni(NO₃)₂·6H₂O, Xilong chemical co., Ltd), cobalt(II) nitrate hexahydrate (Co(NO₃)₂·6H₂O, Tianjin Hebei Sea crystal fine chemical plant), hexamethylenetetramine (HMTA, Afar Sally (Tianjin) chemical co., Ltd), perchloric acid (HClO₄, Tianjin xinyuan chemical co., Ltd), aniline (ANI, Tianjin damao Chemical Reagent Factory), ammonium persulfate (APS, Sigma-Aldrich), and ethanol (CH₃CH₂OH, Its group chemical reagent co., Ltd) were used as received without further purification.

2.2. The synthesis of the NiCo-NPs-in-N/C hybrid structure

NiCo₂O₄ sheets were prepared according to our previous method.^29,30 NiCo₂O₄@PANI core@shell nanosheets were prepared by the one-step polymerization of polyaniline (PANI) on the surface of NiCo₂O₄ (Scheme 1). Typically, 3 mg of NiCo₂O₄ nanosheets was dispersed in 7 mL 1 M HClO₄ aqueous solution by ultrasonication. Then, 0.3 mL of ethanol and 9 μL of 10 mM ANI were injected into the abovementioned mixture at 0 °C to form a uniform solution. After 30 min, 3 mL of 1 M HClO₄ solution containing APS (the molar ratio of ANI/APS was 1 : 1) was added dropwise into the mixed solution and further reacted at 0 °C for 7 h. The obtained product was collected and washed with deionized water and ethanol several times, followed by vacuum-drying at 60 °C. The NiCo-NPs-in-N/C hybrid structure were obtained by carbonization of the as-prepared NiCo₂O₄@PANI core@shell nanosheets at 750 °C for 2 h with a heating rate of 1 °C min⁻¹ under N₂ atmosphere. For comparison, graphene@N-doped carbon core@shell nanoplate (graphene@N/C) was prepared via a similar synthetic protocol but using graphene instead of NiCo₂O₄ sheets and carbonized at 750 °C, and NiCo alloy was also prepared by annealing NiCo₂O₄ nanosheets at 750 °C under reducing atmosphere.

2.3. Preparation of NiCo-NPs-in-N/C modified electrodes

Glass carbon (GC) electrodes (diameter is 3 mm) were carefully polished to a mirror-like plane with 0.3 μm, 0.1 μm and 0.05 μm Al₂O₃ slurries. Then, the electrode were washed thoroughly with excess amounts of water and dried under N₂ gas. The active materials modified GC electrode was prepared by casting 4 μL active materials suspension (1 mg mL⁻¹ NiCo-NPs-in-N/C in N,N-dimethylformamide) on the surface of GC electrode and dried at room temperature. For real sample analysis, no pretreatment process was performed. Human urine was diluted 100 times with 0.1 M PBS (pH = 7.0) to fit the calibration curve and reduce the matrix effect.

2.4. Characterizations

The morphology and structure of the samples were studied by field-emission scanning electron microscopy (FE-SEM (JEORJSM-6700F)) and transmission electron microscopy (TEM (FEI Tecnai G2 20)) with an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) experiments were performed with an ESCA LAB 250 spectrometer using a focused monochromatic Al K_α (hν = 1486.6 eV) X-ray beam with a diameter of 200 μm. X-ray diffraction (XRD) data were obtained on a Y-2000 X-ray diffractometer using copper K_α radiation (λ = 1.5406 Å) at 40 kV, 40 mA. All electrochemical measurements were performed on a CHI660D electrochemical workstation (Chenhua Instrument Company of Shanghai, China). A conventional three-electrode system was used, including a bare or modified GC electrode as working electrode, a platinum wire as an auxiliary electrode, and an Ag/AgCl (saturated KCl) as the reference electrode.

3. Results and discussion

3.1. Characterization of NiCo-NPs-in-N/C

Two dimensional (2D) NiCo-NPs-in-N/C hybrid nanoplates were synthesized through a one-step in situ nanospace confined pyrolysis of as-prepared NiCo₂O₄@PANI core@shell nanosheets (see details in Experimental section). The entire experimental flowchart is schematically illustrated in Scheme 1. First, NiCo₂O₄ sheets were prepared by a hydrothermal method.²⁹ As seen in Fig. S1A & B,† NiCo₂O₄ sheet shows the average size of 2.5 μm in length and 1.5 μm in width with mesoporous structure formed by numerous NiCo₂O₄ nanocrystals, which is in accordance with that observed in the reported literature.²⁹ All the diffraction peaks of the NiCo₂O₄ sheets in the XRD pattern (Fig. S1C†) can be indexed to the cubic spinel NiCo₂O₄ phase (JCPDS card no. 20-0781). Then, NiCo₂O₄ sheets were employed as a planar support to grow PANI by one-pot in situ polymerization of ANI in the presence of APS as the oxidant (the product was noted as NiCo₂O₄@PANI core@shell nanoplates). As shown in Fig. 1, the PANI was uniformly coated on the entire NiCo₂O₄ nanosheets with the ordered PANI nanorod ensembles assembled on the surfaces of the nanosheets, forming a 2D sandwich structure of PANI-NiCo₂O₄-PANI.

Fig. 1 SEM images of NiCo₂O₄@PANI core@shell nanoplates.

The as-prepared NiCo₂O₄@PANI core@shell nanoplates underwent carbonization at a high temperature of 750 °C in N₂ atmosphere for 2 h at a heating rate of 1 °C min⁻¹, allowing partial “escape” of the core from the carbon shell, resulting in the well-distributed NiCo alloy NPs embedded in N-doped carbon nanoplates. Fig. 2 and S2† show the SEM and TEM images of NiCo-NPs-in-N/C nanostructures. The SEM image (Fig. 2A and S2A†) shows that the hybrid structures maintain a 2D planar morphology with N-doped carbon nanorod ensembles on their surface. It can be observed from TEM images (Fig. 2B, C and S2B†) that numerous NPs with a size of 15–20 nm were confined in the hierarchical carbon planar structures. As a further confirmation, the large magnified TEM images in Fig. S3† reveal the light carbon sheath with numerous micropores and a thickness of 3–5 nm around the dark central nanocrystal, indicating the NiCo NPs embedded in the carbon layer. Fig. 2D shows the well-identified interplanar spacing of 0.204 and 0.203 nm for the distances of the (111) and (111) plane of face-centered cubic (fcc) NiCo crystals, respectively.^31–33 The corresponding fast Fourier transformation (FFT) image (inset of Fig. 2D) further confirms the fcc structure of NiCo crystal.

Fig. 2 (A) SEM, (B and C) TEM and (D) HRTEM images of NiCo-NPs-in-N/C (the inset of D shows the corresponding FFT image of NiCo crystal with scale bar being 5 nm⁻¹). (E) XPS spectra of the hybrid structure. Insets of E are the XPS spectra of Co 2p and Ni 2p regions of NiCo-NPs-in-N/C.

The elemental composition and chemical state of NiCo-NPs-in-N/C are characterized by X-ray photoelectron spectroscopy spectrum (XPS) (Fig. 2E). By using a Gaussian fitting method, the Co 2p spectrum can be well fitted with two spin–orbit doublets, characteristic of Co²⁺ and Co³⁺, and two shake-up satellites (identified as “Sat.”).^22,23 A similar case occurred on the Ni 2p spectrum fitting into two spin–orbit doublets, characteristic of Ni²⁺ and Ni³⁺, and two shake-up satellites.^34–36 The Ni/Co molar ratio obtained from the spectrum was found to be close to the initial 1 : 2 Ni : Co molar ratio. About 2.23% nitrogen is introduced into the NiCo-NPs-in-N/C nanostructure.

The temperature-dependent structure change of NiCo-NPs-in-N/C nanostructures under high temperature annealing condition in N₂ was used to study the formation mechanism of NiCo-NPs-in-N/C (Fig. S4†). When the NiCo₂O₄@PANI was carbonized at 350 °C, the NiCo₂O₄ sheets broke into smaller NPs and were simultaneously reduced by carbon precursor and partially converted to metallic NiCo phase (Fig. S4A†). It can be noted that the cubic spinel NiCo₂O₄ phase in the parent nanosheets partially converted into face-centered cubic NiCo alloy (JCPDS cards no. 15-0806 for Co and no. 04-0850 for Ni) at 350 °C (Fig. 3). When the annealing temperature went up to 550 °C, the NiCo-NPs-in-N/C nanostructure was obtained (Fig. S4B†). This is because NiCo₂O₄ was completely reduced to NiCo alloy by carbon at 550 °C, and the nanoscaled metallic NiCo fused and flowed out from the porous carbon shell.³⁷ As seen from the XRD, the samples completely transformed to NiCo alloy and the peak intensity became stronger. With the increase of annealing temperature to 750 °C (Fig. S4C†), the uniform NiCo alloy NPs generally became homogenous and smaller with a size of 15–20 nm. The corresponding XRD result of NiCo-NPs-in-N/C at 750 °C confirms that NiCo alloy NPs are reduced by carbon with increasing carbonization temperature, whereas the NPs agglomerated into large bulks at 950 °C (Fig. S4D†).

Fig. 3 XRD patterns of NiCo₂O₄@PANI core@shell nanoplates carbonization at different temperature: 350, 550, 750, and 950 °C; and the standard XRD patterns of NiCo₂O₄, Ni, and Co.

To reveal the effect of nitrogen functionalities on capacitive performance, the types of N group introduced to NiCo-NPs-in-N/C were further studied by XPS (Fig. S5†). The deconvoluting N 1s region spectrum could provide three type of peaks correlated to different electronic states of nitrogen functional groups: pyridinic (N-6, 398.6 eV), pyrrolic (N-5, 400.0 eV), and quaternary nitrogen (N-Q, 401.1 eV).³⁸ As shown in Fig. S5,† the NiCo-NPs-in-N/C nanostructure from different temperature exists in different electronic states of nitrogen functional groups, which indicates the influence of temperature on the component of materials. Only N-Q and N-6 are dominant in the structure of NiCo-NPs-in-N/C at 750 °C, whereas N-5 is not observed. This could be due to the higher stability of N-Q and N-6 over N-5. These accessible species of N-6 and N-Q may play the important role of improving the electrocatalytic activity of the NiCo-NPs-in-N/C nanostructure.^5,39

Based on the TEM, XRD, and XPS data of the NiCo-NPs-in-N/C at different annealing temperatures, significant optimization of the nanostructure was obtained at 750 °C. The NiCo NPs with uniform size are completely reduced by carbon and confined in the hierarchical carbon shells. Such interesting nanostructure is expected to show the highest electrocatalytic activity over other samples.

In order to verify the effectiveness of NiCo NPs as a new catalysis toward EC determination of AA, DA, and UA, we also prepared 2D N-doped carbon-coated graphene oxide (graphene@N/C) nanoplates through a similar synthetic protocol to NiCo-NPs-in-N/C using graphene oxide instead of NiCo₂O₄ nanosheets and carbonizing at 750 °C. NiCo alloy was also prepared based on the previous literature.⁴⁰ TEM and SEM images of graphene@N/C (Fig. S6†) display that a great number of small humps are uniformly coated on the surface of graphene, indicating the similar nanostructure of graphene@N/C to NiCo-NPs-in-N/C.

3.2. Electrochemical properties

We used Fe(CN)₆^3−/4− as the electrochemical probe to evaluate the electrochemical properties of NiCo-NPs-in-N/C, graphene@N/C and GC electrodes (Fig. 4). As we all know, the peak potential separation (ΔE_p) is the function of the electron transfer rate and the lower the ΔE_p, the higher the electron transfer rate. From the cyclic voltammetry (CV) curves of the samples collected in 5 mM Fe(CN)₆^3−/4− (1 : 1) + 0.1 M KCl solution (Fig. 4A), ΔE_p at the NiCo-NPs-in-N/C electrode is 86.2 mV, which is the lowest ΔE_p. Furthermore, the redox peak current at the NiCo-NPs-in-N/C electrode is higher than the value at graphene@N/C and GC electrodes due to good electronic conductivity. The smaller value of ΔE_p and the higher redox peak currents indicate that the NiCo-NPs-in-N/C electrode exhibits better electrochemical properties than graphene@N/C and GC electrodes.

Fig. 4 (A) CVs and (B) EIS curves obtained for 5 mM Fe(CN)₆^3−/4− (1 : 1) + 0.1 M KCl at bare GC electrode, NiCo-NPs-in-N/C and graphene@N/C electrodes. Scan rate for CVs: 50 mV s⁻¹.

The electron transfer kinetics at different electrodes is also investigated and the corresponding results are shown in Fig. 4B. The distinct semicircular curve at the higher frequency region and linear portion in the low frequency region correspond to the electron transfer (R_ct) and diffusion limited process of the electrochemical reaction, respectively.⁴¹ R_s is the electrolyte resistance between the working and reference electrodes, C is a constant phase element and Z_w is Warburg impedance.^42,43 The impedance diagram was fitted based on the electrical equivalent circulation; see the inset of Fig. 4B. The R_ct value at NiCo-NPs-in-N/C electrode is 49 ohm, which is smaller than graphene@N/C electrode (65 ohm) and GC electrode (345 ohm). The result is in accordance with that observed in Fig. 4A and reconfirms that NiCo-NPs-in-N/C possesses good conductivity and electrochemical properties. All these results imply that the NiCo-NPs-in-N/C is expected to be a promising material for constructing electrochemical biosensors.

3.3. Electrocatalytic oxidation of AA, DA and UA

In order to reveal the electrochemical performance of NiCo-NPs-in-N/C nanostructure towards the oxidation of AA, DA and UA, CV and differential pulse voltammetry (DPV) measurements based on NiCo-NPs-in-N/C, graphene@N/C, NiCo alloy and bare GC electrodes were carried out (Fig. 5). In the case of AA (Fig. 5A), the oxidation peak corresponds to the two electrons and one proton oxidation of hydroxy groups to carbonyl groups in the furan ring of AA (Scheme 2). In detail, the oxidation potential at the NiCo-NPs-in-N/C electrode is more negative than that at the other electrodes, and the corresponding peak current of the NiCo-NPs-in-N/C electrode is higher than that of the graphene@N/C, NiCo alloy and bare GC electrodes, indicating that the electron transfer kinetics of AA oxidation was quicker at NiCo-NPs-in-N/C than for other samples. No peak is observed at the electrodes, which show irreversible electrode processes for AA electro-oxidation.⁴⁴ For DA (Fig. 5B), a couple of redox peaks can be found at every electrode, which corresponds to two-electron oxidation of DA to dopamine quinone and the subsequent reduction of dopamine quinone to DA (Scheme 2). However, a couple of reversible and well-defined redox peaks with a lower ΔE_p of 55 mV can be observed at the NiCo-NPs-in-N/C electrode, indicating better reversibility for the redox process, while the NiCo-NPs-in-N/C electrode has a much stronger response to the oxidation of DA compared to graphene@N/C, NiCo alloy and bare GC electrodes. Fig. 5C shows the oxidation behavior of UA, the CV curves reveal that UA is first oxidized to quinonoid, and then undergoes a rapid chemical reaction, which is considered an EC mechanism (Scheme 2).⁴⁵ NiCo-NPs-in-N/C electrode exhibits the most negative oxidation peak potential and a significant enhancement in the oxidation current when compared to that of graphene@N/C, NiCo alloy and bare GC electrodes. These results reveal a fact that NiCo-NPs-in-N/C possesses the highest activity to graphene@N/C, NiCo alloy and bare GC.

Fig. 5 CVs of bare GC electrode, NiCo-NPs-in-N/C, NiCo alloy, and graphene@N/C electrodes in 0.1 M PBS (PH 7.0) containing (A) 1.0 mM AA, (B) 1.0 mM DA, (C) 1.0 mM UA, and (D) 1.0 mM AA + 0.05 mM DA + 0.1 mM UA.

Scheme 2 Electrochemical oxidation of AA, DA and UA.

In order to study the electrocatalytic properties and selectivity of NiCo-NPs-in-N/C for the simultaneous determination of UA, AA and DA, the DPV response of NiCo-NPs-in-N/C, graphene@N/C, NiCo alloy and bare GC electrodes in the co-existence system of AA, DA and UA was also collected (Fig. 5D). The NiCo-NPs-in-N/C electrode exhibits the highest activity, even higher than graphene@N/C and NiCo alloy. Typically, the separated voltammetric peaks between AA–DA, DA–UA, and AA–UA at the NiCo-NPs-in-N/C are up to 178, 122, and 300 mV, respectively. In contrast, the oxidation peaks of AA and DA overlap with each other at the graphene@N/C electrode and NiCo alloy, and the DPV obtained at the bare GC electrode exhibits inconspicuous peaks. Therefore, NiCo-NPs-in-N/C nanostructure holds superior electrocatalytic activity and selectivity to graphene@N/C, NiCo alloy and bare GC electrodes.

Based on CVs and DPV data, the excellent electrocatalytic properties and perfect selectivity of the NiCo-NPs-in-N/C electrode is probably attributed to the synergistic effect of NiCo alloys, nitrogen groups and the hierarchical structures. Moreover, the interactions between NiCo-NPs-in-N/C layers and these biomolecules can promote the charge transfer of three molecules. More significantly, the uniform NiCo alloy NPs play a critical role in the high catalytic activity toward to the oxidation of AA, DA and UA.

3.4. Effects of scan rate

To investigate the reaction kinetics and the influence of scan rate on the CV responses of AA, DA and UA at the NiCo-NPs-in-N/C electrode was investigated. As shown in Fig. S7,† the oxidation peak currents of AA, DA and UA as a function of the square root of scan rate is in the range of 5–500 mV s⁻¹, which indicates that electrochemical oxidation is a diffusion-controlled process.

3.5. Simultaneous determination of AA, DA and UA

Since DPV technique has much higher current sensitivity and better resolution compared to CV technique, simultaneous detection of UA, AA and DA was carried out using DPV method. As shown in Fig. 6, a series of DPV curves are obtained by changing the concentration of one biomolecule while keeping the other two biomolecules at a constant value. Three well-defined oxidation peaks were observed and their currents increased, whereas the oxidation potential remained steady. The electrochemical response of AA increases linearly with the increase of its concentration in the range of 50–1500 μM, and the corresponding linear function is I_AA (μA) = 0.13075 + 0.00247C_AA (μM) with the correlation coefficient of R = 0.9952. The oxidation peak current of DA increases with the increase of DA concentration from 0.5 to 900 μM. The linear regression equation is calibrated as I_DA (μA) = 0.1313 + 0.0212C_DA (μM) with the correlation coefficient of R = 0.9945. Similarly, the peak current of UA increases linearly with the increase of the UA concentration in the range of 10–500 μM with the linear function I_UA (μA) = 0.13281 + 0.01399C_UA (μM) with correlation coefficient of R = 0.9983. Detection limits for AA, DA and UA are 0.091 μM, 0.080 μM and 0.014 μM, respectively, at S/N = 3. We further compared the catalytic performance of our NiCo-NPs-in-N/C with various reported metal–carbon-based catalyst (Table S1†). Obviously, NiCo-NPs-in-N/C exhibit excellent catalytic performance with the lowest detection limit and widest linear range.

Fig. 6 DPVs at the NiCo-NPs-in-N/C electrode in 0.1 M PBS (PH 7.0) (A) containing 80 μM DA, 160 μM UA and different concentrations of AA from 0 to 1500 μM; (B) containing 1000 μM AA, 160 μM UA and different concentrations of DA from 0 to 900 μM; (C) containing 1000 μM AA, 80 μM DA and different concentrations of UA from 0 to 500 μM. Insets: plots of I (peak) vs. concentration for AA, DA and UA, respectively.

3.6. Stability and reproducibility

The stability and reproducibility of NiCo-NPs-in-N/C nanosheets electrode was also investigated in the mixture solution containing 1.0 mM AA, 0.05 mM DA and 0.1 mM UA. Five electrodes modified by NiCo-NPs-in-N/C were used to measure the three target molecules with DPV method; the current response remained almost constant and the relative standard deviations (R. S. D.) were 3.5%, 2.9%, 3.7% for AA, DA and UA, respectively. Current signals of the electrode also showed no obvious decreases (4.6%, 3.9%, and 4.5% for 1.0 mM AA, 0.05 mM DA and 0.1 mM UA, respectively), relative to the initial response after storage in air at room temperature for a month. It can be speculated that the carbon shell around the NiCo alloy NPs in NiCo-NPs-in-N/C acts as an effective protection for the NiCo NPs from dissolution in corrosive conditions, thus leading to excellent stability and reproducibility.

3.7. Analysis of real samples

To illustrate the feasibility of NiCo-NPs-in-N/C electrode for the simultaneous determination of AA, DA and UA in biological fluids, the electrode was applied to select the three molecules in human urine. The human urine sample was diluted 100 times with 0.1 M PBS before the measurement, and certain amounts of AA, DA and UA were added into the diluted sample. Spike recoveries were 98.7%, 102.3%, 101.6% for 1.0 mM AA, 0.05 mM DA and 0.1 mM UA (Table S2†), respectively, implying NiCo-NPs-in-N/C has great potential for simultaneous determination of AA, DA and UA in real samples.

The results from the electrocatalytic studies toward simultaneous determination of AA, DA and UA indicate that the confinement effect of NiCo NPs and the hierarchical structure of N-doping carbon play important roles in optimizing the electrocatalytic properties of NiCo-NPs-in-N/C. We found that such particular NiCo-NPs-in-N/C plate architectures can well address several important challenging issues related to the electrocatalyst: (a) the synergistic effect of the bimetallic system encourages the electrocatalytic property of NiCo NPs in NiCo-NPs-in-N/C. (b) N-doped carbon shell can act as an armour or buffer for protecting the NiCo NPs from detachment, dissolution, migration, and aggregation after long-term use. (c) The 2D hierarchical structure coupled with N-doped functionalities with high electron conductivity provides perfect pathways for electrons and mass transport, thus accelerating the charge transform kinetics of the target molecules at the electrode.^46,47 Accordingly, it is shown that the catalytic performance of our NiCo-NPs-in-N/C is superior to most reported metal–carbon-based catalysts (Table S1†). Taking their robust electrocatalytic performance into account, we conclude that rationally designed architectures of the metal–carbon-based catalytic can significantly realize the optimization of the electrocatalytic performance for biosensor.

4. Conclusions

In conclusion, an intriguing composite of sandwich-structured NiCo nanoparticle-N-doped carbon nanoplate was facilely synthesized in a one-step in situ nanospace confined pyrolysis strategy, and exhibited great potential for the simultaneous determination of AA, DA and UA. In such a structure, homogeneous NiCo NPs (15–20 nm) are embedded in the compartment of carbon nanoplates. Initial study on the formation mechanism of NiCo-NPs-in-N/C was carried out through changing the annealing temperature. The control experiments reveal that proper annealing temperature is the key to in situ formation of well-defined NiCo-NPs-in-N/C nanoplates. Significantly, electrochemical features could be efficiently regulated by the confinement effect of NiCo NPs and the hierarchical structure of N-doping carbon for NiCo-NPs-in-N/C. As a consequence, the advanced overall performance has been optimized for NiCo-NPs-in-N/C, which exhibits excellent catalytic activity towards the electrooxidation of AA, DA, and UA. Therefore, NiCo-NPs-in-N/C nanoplates show well-separated voltammetric peaks between AA and DA and DA and UA at the NiCo-NPs-in-N/C up to 178 and 122 mV, respectively. For simultaneous sensing of three analytes, the NiCo-NPs-in-N/C nanoplates exhibit the widest linear response range for AA, DA and UA of 500–1500, 0.5–900 and 10–500 μM with lowest detection limits of 0.091, 0.080, and 0.014 μM, respectively, which is even better than ever reported relevant catalysts. In addition, NiCo-NPs-in-N/C also exhibits good reproducibility and stability. Therefore, we expect that the new concept of confining metal NPs in carbon architecture can be extended to produce various inorganic NPs–carbon based materials for catalytic and other applications.

Acknowledgements

This study was financially supported by the National Natural Science Foundation of China (Nos 21101141 and 51173170), the Program for New Century Excellent Talents in Universities (NCET), and the Open Project Foundation of Key Laboratory of Advanced Energy Materials Chemistry of Nankai University (2015-32).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra10937j

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