Novel bimetallic Cu/Ni core-shell NPs and nitrogen doped GQDs composites applied in glucose in vitro detection

In present work, a highly sensitive biosensor with high selectivity for glucose monitoring is developed based on novel nano-composites of nitrogen doped graphene quantum dots (N-GQDs) and a novel bimetallic Cu/Ni core-shell nanoparticles (CSNPs) (Cu@Ni CSNPs/N-GQDs NCs). With the tuned electronic properties, N-GQDs helped bimetallic core-shell structure nanomaterials from aggregation, and separate the charges generated at the interface. This novel nano-composites also have the good electrical conductivity of N-GQDs, catalyst property of Cu/Ni bimetallic nano composite, Cu@Ni core-shell structure and the synergistic effect of the interaction between bimetallic nano composite and N-GQDs. While modified the electrode with this novel nano-composites, the sensor’ linear range is 0.09 ~ 1 mM, and the limit of detection (LOD) is 1.5 μM (S/N = 3) with a high sensitivity of 660 μA mM-1 cm-2, and rapid response time (3 s). Its’ LOD is more than 74 times lower than the traditional Cu@Ni CSNPs modified working electrode. It also has higher sensitivity and wider linear range. This indicates the great potential of applying this kind of nano composites in electrode modification.


Introduction
With the rapid development of nanotechnology, a lot of carbon materials have emerged in recent years, such as fullerenes, multi-wall carbon nanotubes, carbon nanofibers, graphene [1], graphene quantum dots (GQDs) and so on. These carbon materials show unique physical and chemical properties. Among these materials, GQDs [2,3] are graphene sheets with only monolayer or less layers. They are a kind of zero dimensional carbon materials with sizes less than 10nm. They usually contain functional groups, e.g. carboxyl, hydroxyl, carbonyl, epoxide etc. at the edges [4]. They were popular reported for their high biocompatibility, small sizes and low costs. Quantum confinement and edge effects endow them with optical activity, conductivity and chemical inertness. GQDs exhibit strong photoluminescence (PL), excellent a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 electrochemiluminescence (ECL) and electrochemical activity because they are favorable electron donors and acceptors due to their large surface area and abundant edge sites. Surface chemistry also applied to GQDs synthesis and makes the interesting properties available.
Owing to these properties, GQDs have gained wide attention for their enormous potential in varies applications. For instance they are widely applied in photovoltaics, organic light emitting diodes, fuel cells, photocatalysis, bioimaging, biosensing, biomedicine, environmental monitoring, thermal interface materials etc [5][6][7][8]. Biosensor is one of the most interesting application [8][9][10][11][12]. Combining with abundant detection methods, scientists designed varies analytical strategies based on GQDs' unique properties. Among all the biosensors, glucose sensors, especially glucose electrochemical sensors, are widely studied for very long history [13][14][15][16]. Because glucose is a very important carbohydrate and play irreplaceable role for organisms. It is also the index for diabetes which is one of the most common deadliest diseases and it is affecting by several millions of people all over the world.
In this work, the novel bimetallic Cu/Ni and N-GQDs nano-composites (Cu@Ni CSNPs/ N-GQDs NCs) have been synthesized by hydrothermal method and a one-pot solvothermal method. With our gentle synthesis method, the size of N-GQDs can be uniform with the nitrogen content about 12.93%. The size of Cu @ni CSNPs/N-GQDs NCs is 30-80 nm, and N-GQDs is uniformly coated on its surface, with bimetallic Cu/Ni co-catalytic performance and good electrical conductivity. A series of non-enzymatic glucose sensors are constructed with Cu@Ni CSNPs/N-GQDs NCs modified glassy carbon electrode (Cu@Ni CSNPs/ N-GQDs/GCE), Cu@Ni CSNPs/GCE and Cu@Ni CSNPs/N-GQDs/GCE. Their electrochemical properties and electrocatalytic activities are compared. It is excited that Cu@Ni CSNPs/ N-GQDs/GCE shows the best electrocatalytic performance for glucose oxidation, and displays the lowest detection limit(LOD) 1.5 μM (S/N = 3), a wider linear range from 0.09 mM to 1 mM and high sensitivity 660 μA mM -1 cm -2 . Comparing with the sensor based on Cu@Ni CSNPs modified working electrode, the electrode modified with Cu@Ni CSNPs/ N-GQDs NCs makes the sensor's LOD more than 74 times lower, higher sensitivity and wider linear range. These indicate that well designed N-GQDs' composites have great application prospects in improving electron migration rate and electrocatalytic performance of electrode surface.

Preparation of N-GQDs
N-GQDs were synthesized by hydrothermal method. 1g citric acid and 0.947 g urea weretaken into the Teflon-lined, 10 ml ultrapure water was added and stirred until citric acid and urea fully dissolved. Then put the stainless-steel autoclave into the muffle furnace and heated at 180˚C for 5 hours. After the sample was cooled to room temperature, the mixture was filtered and dialyzed against a 3000 D dialysis bag to neutrality, then the solution was dried by a freeze dryer to obtain the powder.

Preparation of Cu@Ni CSNPs/N-GQDs NCs
The solution contained 0.0335 g N-GQDs and 33.3 ml EG was dissolved and ultrasonically dispersed 5 hours. The solution contained 0.1482 g CuCl 2 �2H 2 O and 0.2 g NiCl 2 �6H 2 O and 33.3 ml EG and the solution contained 0.675 g NaOH and 8.3 ml EG were both prepared by ultrasonically dispersion for 1 hour. Then the solution of CuCl 2 �2H 2 O and NiCl 2 �6H 2 O was added dropwise into the N-GQDs solution under stirring, following with the NaOH solution added dropwise and stirred at room temperature for 1 hour. When the reaction was thoroughly finished, the mixture was transferred to a Teflon-lined and heated at 200˚C in the muffle furnace for 5 hours. After the sample was cooled to room temperature, it was stirred for 10 minutes and centrifugalized at 8000 rpm for 5 minutes, then washed repeatedly with ethanol and ultrapure water 3 times and vacuum dried overnight to obtain the black composite powder.

Preparation of Cu@Ni CSNPs, Cu NPs and Ni NPs
For convenience of comparison, Cu@Ni CSNPs, Cu NPs and Ni NPs were prepared respectively with the same procedure. 0.2557 g CuCl 2 �2H 2 O and 0.3565 g NiCl 2 �6H 2 O and 60 ml EG were dissolved and ultrasonically dispersed for 1 hour. Then the NaOH solution mentioned was added dropwise into the above solution, and stirred at room temperature for 1 hour. While the reaction thoroughly finished, the mixture was heated at 200˚C in the muffle furnace for 5 hours. After the mixture was cooled at room temperature, it was stirred for 10 minutes and centrifugalized at 8000 rpm for 5 minutes, then washed repeatedly with ethanol and ultrapure water respectively. Finally, the mixture was vacuum dried overnight to obtain the black Cu@Ni CSNPs powder. By the same conditions, the Cu NPs and Ni NPs were also prepared.

Preparation of the modified GCE
40 g DMF and 4 g Nafion solvent were taken and ultrasonically dispersed for 1 hour. 0.02 g Cu@Ni CSNPs/N-GQDs NCs was dispersed into 20 ml DMF and Nafion mixture, then ultrasonically dispersed for 1 hour to obtain catalyst suspension. After the GCE preparation, 10 μL catalyst suspension was coated on the working electrode. Then the electrode dried under infrared lamp to obtain the modified Cu@Ni CSNPs/N-GQDs/Nafion/GCE. For comparison, the Cu@Ni CSNPs/Nafion/GCE was also prepared by the same method.

Materials characterization
The morphology of N-GQDs, Cu@Ni CSNPs and Cu@Ni CSNPs/N-GQDs NCs composites were characterized by TEM and HRTEM. As can be seen from Fig 1A, N-GQDs are "point" shaped and evenly distributed. In Fig 1B, HRTEM image shows that the N-GQDs have average diameter about 5 nm, and the lattice spacingis about 0.24 nm, corresponding to the (100) lattice spacing of the GQDs [40]. Fig 1C is the TEM image of Cu@Ni CSNPs. Because of their double alloy structure, high crystallinity can be clearly seen from the figure. They have size about 50-110 nm, and the average lattice spacing 0.203 nm (Fig 1D), corresponding to the {111} crystal plane of Cu and Ni [1]. Fig 1E is the TEM image of Cu@Ni CSNPs/N-GQDs NCs.

Electrochemical behavior of Cu@Ni CSNPs/Nafion/GCE and Cu@Ni CSNPs/N-GQDs/Nafion/GCE
The modified Cu@Ni CSNPs/Nafion/GCE and Cu@Ni CSNPs/N-GQDs/Nafion/GCE electrodes were placed in cell containing 0.1 M NaOH solution to be tested at different scan rates. As shown in Fig 3A, Fig 3D. Both of them indicate that the electrochemical kinetics of these two electrodes are surface controlled. Compared with these two electrodes, modified Cu@Ni CSNPs/N-GQDs/Nafion/GCE electrode has better linearity and better electrochemical performance. As the scan rate increasing, the anodic peak potential is positive and the cathodic potential is negative, that caused a larger peak-to-peak separation. These results are due to nucleation of NiO(OH) and increased activity centers of Ni 3+ and Ni 2+ species [41,42].

Electrocatalytic oxidation of Glucose on Cu@Ni CSNPs/Nafion/GCE and Cu@Ni CSNPs/N-GQDs/Nafion/GCE
CVs was used to study the relationship between current and glucose concentration. The CVs was obtained by placing modified electrodes in solutions containing different glucose concentrations at scan rate of 100 mV s -1 . In Fig 4A, the CVs of modified Cu@Ni CSNPs/Nafion/ GCE electrode has a glucose concentration range from 0.1 μM to 1 mM. Both anodic and cathodic currents are increased with the increasing of glucose concentration. Fig 4B shows the linear relationship between glucose concentration and current, Ipa = -0.00127 c-2.12802 (R 2 = 0.95372), Ipc = 2.34641 ×10 −5 c + 0.59356 (R 2 = 0.63127). In Fig 4C, the CVs of the modified Cu@Ni CSNPs/N-GQDs/Nafion/GCE electrode has a glucose concentration range from 0.1 μM to 1 mM, both anodic and cathodic currents are increased with the increasing of glucose concentration. Fig 4D shows  when the scan rate increasing, the current gradually increasing. The anodic potential range is from +0.5~0.7 V, which is the constant potential range for the next i-t curve test.
Under the optimal conditions, +0.6 V is selected as the constant potential in the range of anodic potential of +0.5~0.7 V. In Fig 6A-6D they show the current-time responses at +0.6 V with an increasing glucose concentration every 50 s for the Cu@Ni CSNPs/Nafion/GCE and Cu@Ni CSNPs/N-GQDs/Nafion/GCE, and the linear relationship between the catalytic current and glucose concentration. As shown in Fig 6A, it is the i-t curve of Cu@Ni CSNPs/ Nafion/GCE at different glucose concentrations. The linear relationship between current and glucose concentration is shown in Fig 6B. The current response of the sensor exhibits a linear dependence on glucose concentration from 0.2 mM to 1 mM (i = 0.0853 c-0.00018, R = 0.99973). The detection limit of glucose using Cu@Ni CSNPs/Nafion/GCE is found to be 111.4 μM (S/N = 3) with the sensitivity of 85.3 μA mM -1 cm -2 . As shown in Fig 6C, it is the i-t Novel bimetallic Cu/Ni core-shell NPs and nitrogen doped GQDs composites applied in glucose detection curve of Cu@Ni CSNPs/N-GQDs/Nafion/GCE at different glucose concentrations. The linear relationship between current and glucose concentration is shown in Fig 6D. The current response of the sensor exhibits a linear dependence on glucose concentration from 0.09 mM to 1 mM (i = 0.66 c + 0.125, R = 0.99952). The detection limit of glucose using Cu@Ni CSNPs/ N-GQDs/Nafion/GCE is found to be 1.5 μM (S/N = 3) with the sensitivity of 660 μA mM -1 cm -2 . From this, it can be clearly concluded that Cu@Ni CSNPs/N-GQDs/Nafion/GCE is more sensitive to glucose determination. This is because GQDs itself is conductive, and N-GQDs has more hole electron pairs due to nitrogen atoms have been successfully doped, which greatly improve the electrical conductivity. Glucose is catalyzed by Cu and Ni in the composites, and N-GQDs are coated on the surface of Cu@Ni CSNPs, which improves the electron mobility between the electrode and the electrolyte, so that the working electrode to Novel bimetallic Cu/Ni core-shell NPs and nitrogen doped GQDs composites applied in glucose detection detect glucose within a very short time, which greatly improves the sensitivity of the electrode and reduces the detection limit, providing conditions for real-time, rapid and accurate determination of glucose concentration.

Selectivity of glucose by Cu@Ni CSNPs/N-GQDs/Nafion/GCE
The anti-interference property and selectivity for glucose determination are crucial in the development of glucose biosensors. Chemical species such as uric acid (UA), dopamine (DA), ascorbic acid (AA), and NaCl that easily oxidize are always present with glucose in human blood. In this study, interference experiments were detected by adding 0.1 mM interference component to a 0.1 M NaOH solution containing 0.5 mM glucose. As shown in S4A and S4B  Novel bimetallic Cu/Ni core-shell NPs and nitrogen doped GQDs composites applied in glucose detection

Summary
In summary, Cu@Ni CSNPs and Cu@Ni CSNPs/N-GQDs NCs were successfully synthesized by one-pot solvothermal method. Our experiments show that the electrochemical response of Cu@Ni CSNPs/N-GQDs/Nafion/GCE to glucose determination was the highest. Cu@Ni CSNPs/N-GQDs/Nafion/GCE has the advantages of low cost, high sensitivity and good selectivity. One more advantage of Cu@Ni CSNPs/N-GQDs/Nafion/GCE is its' wide linear range is 0.09~1 mM. And the detection limit is 1.5 μM (S/N = 3), high sensitivity of 660 μA mM -1 cm -2 , rapid response time (3 s). Comparing with the sensor based on Cu@Ni CSNPs modified working electrode, this novel nano-composite of Cu@Ni CSNPs/ N-GQDs NCs makes the sensor's LOD more than 74 times lower, also has higher sensitivity and wider linear range. Furthermore, the interference components show insignificant interference in determination of glucose, and Cu@Ni CSNPs/N-GQDs/Nafion/GCE has high selectivity for glucose determination. The results indicate that the biosensor based on Cu@Ni CSNPs/N-GQDs/Nafion/GCE has potential application prospect in the determination of glucose, the application of GQDs in biosensor has a great prospect. It also indicates the great potential to apply this kind of nano composites in electrode modification and high sensitivity biomolecule detection.
Supporting information S1 Table.