Tuning Co/Ni Ratio in Co–Ni Bimetallic Hybrid for Electrochemical Detection of Glucose

: Transition metallic binary alloys have attracted enormous attention in regard to the non-enzymatic detection of glucose due to their high electrocatalytic activities induced by the synergistic effect between the individual metallic species. However, the easy aggregation of the bimetallic particles has limited their performance. Herein, a facile metal–organic framework (MOF)-derived strategy is developed to synthesize a hybrid containing binary Co–Ni nanoparticles decorated on an N-doped porous carbon matrix (Co x Ni y /N-C) for the non-enzymatic detection of glucose. The Co/Ni ratio in the hybrid is investigated to regulate its electrocatalytic behaviors for glucose sensing. A hybrid with the optimal Co/Ni ratio of 1:1 displays two linear detection ranges (0.5 µ M to 1 mM and 1 mM to 10 mM) with a detection limit of 0.11 µ M for glucose. The feasibility of using this hybrid-modified SPE for glucose detection in real serum samples has also been validated.


Introduction
As one of the most popular chronic diseases, diabetes constitutes a great threat to human health.The number of diabetes patients worldwide is expected to reach 642 million by 2040 [1].Abnormal glucose levels in the body fluids of patients with diabetes usually increase their risk of diabetes complications, such as blindness, heart disease, kidney failure, and foot damage [2].Therefore, the effective and accurate detection of glucose levels is crucial for patients with diabetes.
The enzyme-based electrochemical method is a widely used method for glucose detection.However, it suffers from complicated enzyme immobilization procedures and the poor stability of the enzyme.To overcome these limitations, non-enzymatic electrochemical methods have attracted much attention in recent years [3].Transition metals, such as Co, Ni, and their alloys, have been widely investigated as the catalysts for non-enzymatic glucose sensing due to their low cost and excellent catalytic activities [3][4][5][6][7][8][9].Among these materials, Co-Ni bimetallic alloys have received particular attention for glucose detection due to their synergistic effect on the electro-oxidation of glucose, as well as their tunable electronic and geometric structures [2,[10][11][12][13][14].The rich valence states provided by Co-Ni alloys can offer more active sites than their individual species [13].However, the easy agglomeration of as-synthesized Co-Ni alloys has severely limited their catalytic performance [14].Therefore, it is highly desirable to develop a method to prepare a Co-Ni alloy with an optimal microstructure to achieve high electrocatalytic efficiency.
In recent years, metal-organic frameworks (MOFs) with tunable microstructures and compositions have been explored as the templates for synthesizing porous carbon-based hybrids with various microstructures using the pyrolysis process [15].The as-formed porous carbon matrix exhibits an enhanced electrical conductivity, which is beneficial for the charge transfer during the electrocatalytic process [16,17].At the same time, the presence of heteroatoms in the carbon substrate induced by the pyrolysis of the organic ligands can retain the dispersed metal nanoparticles on the substrate and prevent their aggregation, offering abundant active sites for mass transport and electrocatalytic activity [18,19].Recent studies have shown that the proportion of Co and Ni in the Co-Ni alloy plays an important role in determining its sensing performance for glucose [20][21][22][23][24][25][26].The charge migration and electronic properties of the active sites in the alloy can be modulated by tailoring the Co/Ni ratio, thereby influencing its electrocatalytic performance [21].
Herein, we develop a facile method to prepare a bimetallic Co-Ni hybrid for the detection of glucose.The hybrid containing Co-Ni alloy nanoparticles decorated on a porous N-doped carbon matrix (CoxNiy/N-C) is synthesized through the one-step pyrolysis of the MOF precursor.The effects of the Co/Ni ratio in the hybrid on the microstructure and electrocatalytic performance toward glucose are investigated.A hybrid with the optimal Co/Ni ratio shows outstanding performance toward the electro-oxidation of glucose due to the synergetic effect between the Co and Ni species and the presence of the N-dopant in the carbon matrix.This hybrid is also successfully used to determine the glucose levels in real serum samples.

Synthesis of Co x Ni y /N-C hybrids
The metal salt aqueous solution was prepared by dissolving a total 6.4 mM of Co(NO 3 ) 2 •6H 2 O and Ni(NO 3 ) 2 •6H 2 O mixture with different molar ratios (3:1, 1:1 and 1:3) in a 16 mL aque- ous solution containing 4 mM Zn(NO 3 ) 2 •6H 2 O.The 16 mL of the as-prepared metal salt solution was then mixed with 160 mL of 3.45 M 2-methylimidazole aqueous solution under magnetic stirring at room temperature for 6 h to obtain ZnCoNi-MOF solution.Subsequently, the ZnCoNi-MOF solution was centrifuged at a speed of 10 k rpm for 20 min and washed three times with deionized water, followed by a freeze-drying process for 24 h to obtain ZnCoNi-MOF powder.The as-received ZnCoNi-MOF powder was then annealed at 900

Preparation of Hybrid-Modified Electrodes
Three-electrode system-based screen-printed electrodes (SPEs, Yituo Biosensing Technology, Suzhou, China) were employed as the substrate to investigate the sensing performance of the hybrids for glucose.In bare SPEs, the carbon electrode, Ag/AgCl electrode and carbon electrode were used as the working electrode, reference electrode and counter electrode, respectively.The hybrid ink solution was prepared by dissolving 2 mg hybrid powder into 1 mL isopropanol solution containing 0.5 wt% Nafion.For the modification of the carbon-based working electrode, 5 µL of the hybrid ink was dropped on the working electrode with an area of 3 mm 2 and dried at room temperature for 2 h to achieve the hybrid-modified SPE.

Characterizations
The morphology and elemental composition of the hybrids were characterized using a field emission scanning electron microscope (SEM, Quattro, 20 kV) and atransmission electron microscope (TEM, JEM-2100F, 200 kV), respectively.X-ray diffraction (XRD, Rigaku D/Max 2500, Tokyo, Japan) was used to determine the crystal structure of the samples using Cu Kα radiation at 40 kV.The N 2 adsorption-desorption isotherms were measured using a Quadrasorb analyzer at 77 K after the pretreatment of the sample in a vacuum at 473 K for 2 h.The specific surface areas of the hybrids were calculated using the Brunauer-Emmett-Teller (BET) method.

Electrochemical Measurements
Cyclic voltammetry (CV) and amperometry measurements (i-t) were employed to evaluate the electrochemical performance of the hybrid-modified SPEs, which were performed in an electrochemical cell containing 100 mL 0.1 M NaOH aqueous solution using CHI660E electrochemical workstation from Shanghai Chenhua Instrument Co., Ltd.(Shanghai, China).For the i-t measurement, different concentrations of glucose were successively added into a 0.1 M NaOH aqueous solution every 50 s at the applied potential of 0.6 V.

Morphology and Structure Characterization
The hybrids were obtained through the pyrolysis of MOF precursors at 900 • C for 2 h under N 2 gas.During the calcination process, the Zn species were evaporated and the organic linkers containing a rich nitrogen source were carbonized, forming an N-doped porous carbon substrate decorated with metallic particles [27].The Co/N-C and Ni/N-C hybrids were synthesized for comparison.The morphologies of the as-prepared hybrids were characterized by SEM.As shown in Figure 1a,b, the Co/N-C and Ni/N-C hybrids exhibit a porous structure decorated with particles of irregular morphologies and tubular structures.For Co 3 Ni 1 /N-C and Co 1 Ni 1 /N-C hybrids, uniformly distributed spherical particles are formed on the substrate (Figure 1c,d).However, after a further increase in the Ni content to a Co/Ni ratio of 1:3, the particles show irregular spherical shapes and aggregation (Figure 1e).We further calculated the specific surface areas of the hybrids using the BET method (Figure S1 and Table S1).Among these hybrids, the Co 1 Ni 1 /N-C hybrid displays the highest specific surface area of 375.64 m 2 /g, beneficial for mass transport [21].The TEM and the corresponding EDS elemental mapping images confirm the homogenous distribution of C, N, O, Co, and Ni elements in the Co x Ni y /N-C hybrids (Figures 1f and S2).The uniform distributions of Ni and Co on the carbon matrix at the same location in the hybrids support the formation of the Co-Ni binary alloy on the carbon matrix.
The crystalline structures of the as-prepared hybrids were analyzed using XRD.The XRD patterns of Co x Ni y /N-C hybrids are similar to those of the Co/N-C and Ni/N-C hybrids (Figure 2a).The relative broad peak at 26 • was assigned to the (002) plane of graphitic carbon (JCPDS No. 89-8487) [20].There are three diffraction peaks at around 44 • , 52 • , and 76 • , which are indexed to the (111), (200), and (220) planes of the facecentered cubic (FCC) structures of Co (JCPDS No. 04-0850) and Ni (JCPDS No. 04-0850), respectively [28].No other obvious peaks are observed, suggesting the evaporation of the Zn species during the pyrolysis process.Figure 2b shows the zoomed-in (111) peak of the hybrids.Compared with pristine Co/N-C, the zoomed-in (111) peak of the Co x Ni y /N-C hybrid gradually shifts towards a higher 2θ value with the increase in the Ni amount.This is due to the lattice contraction of the Ni alloying with Co, confirming the formation of the Co-Ni alloy [20,29].No other planes were observed for the Co x Ni y /N-C hybrids, implying that the formation of the Co-Ni alloy has not destroyed the crystal structure of Co or Ni due to the similar lattice parameters of Co and Ni.The crystalline structures of the as-prepared hybrids were analyzed using XRD.The XRD pa erns of CoxNiy/N-C hybrids are similar to those of the Co/N-C and Ni/N-C hybrids (Figure 2a).The relative broad peak at 26° was assigned to the (002) plane of graphitic carbon (JCPDS No. 89-8487) [20].There are three diffraction peaks at around 44°, 52°, and 76°, which are indexed to the ( 111    The crystalline structures of the as-prepared hybrids were analyzed using XRD.The XRD pa erns of CoxNiy/N-C hybrids are similar to those of the Co/N-C and Ni/N-C hybrids (Figure 2a).The relative broad peak at 26° was assigned to the (002) plane of graphitic carbon (JCPDS No. 89-8487) [20].There are three diffraction peaks at around 44°, 52°, and 76°, which are indexed to the (111), (200), and (220) planes of the face-centered cubic (FCC) structures of Co (JCPDS No. 04-0850) and Ni (JCPDS No. 04-0850), respectively [28].No other obvious peaks are observed, suggesting the evaporation of the Zn species during the pyrolysis process.Figure 2b    The chemical compositions and valence states of the elements in the hybrids were characterized using XPS (Figures 2c-f and S3).Three peaks in the high-resolution C 1s spectra located at 284.8 eV, 285.6 eV, and 289.1 eV, are assigned to C=C, C-N and O-C-O, respectively (Figure 2c) [30].The existence of C=C and C-N indicates the graphite carbon structure and the doping of N in the carbon matrix during the pyrolysis of the MOF precursors, respectively [30].For the N 1s spectra, four peaks at 398.9 eV, 400.6 eV, 401.4 eV, and 403.4 eV are ascribed to pyridinic-N, pyrrolic-N, graphitic-N, and oxidized-N, respectively (Figure 2d) [31].It has been reported that the presence of pyridinic-N and pyrrolic-N can provide active sites and increase the charge density for electrocatalytic reactions by tailoring the electronic properties of the carbon substrate with their lone pair of electrons [32][33][34] S2).Meanwhile, it has been shown that the existence of the N dopant can prevent the aggregation of the metallic particles [19].Figures 2e and S3 2f and S3) [35,36].The peaks attributed to Ni 2p 3/2 (~855.0eV), Ni 2p 1/2 (~872.6 eV) and two satellites (~860.3eV and ~879.1 eV, respectively) are Ni 2+ [15].The presence of Co 0 and Ni 0 confirms the reduction of Co 2+ and Ni 2+ species during the pyrolysis, while the existence of Co 2+ and Ni 2+ in the hybrids indicates the surface oxidation by the atmosphere [29,37].tailoring the electronic properties of the carbon substrate with their lone pair of electrons [32][33][34].The amount of pyridinic and pyrrolic N in the Co1Ni1/N-C hybrid (72.1%) is the highest among these hybrids (68.3% for Co/N-C, 69.5% for Ni/N-C, 71.4% for CO3Ni1/N-C, and 70.1% for CO1Ni3/N-C, respectively) (Table S2).Meanwhile, it has been shown that the existence of the N dopant can prevent the aggregation of the metallic particles [19].Figure 2e and Figure S3 reveal that the Co 2p spectra of the CoxNiy/N-C and Co/N-C hybrids compose the two main peaks of Co 2p3/2 and Co 2p1/2, which are further deconvoluted into four subpeaks, corresponding to Co0 (~778.5 eV and ~794.1 eV) and Co 2+ (~780.4eV and ~797.0 eV), and two satellites at around 784.4 eV and 804.2 eV [15].The Ni 2p spectra of the CoxNiy/N-C and Ni/N-C hybrids have confirmed the presence of metallic Ni at around 852.8 eV and 870.3 eV, respectively (Figures 2f and S3) [35,36].The peaks attributed to Ni 2p3/2 (~855.0eV), Ni 2p1/2 (~872.6 eV) and two satellites (~860.3eV and ~879.1 eV, respectively) are Ni 2+ [15].The presence of Co 0 and Ni 0 confirms the reduction of Co 2+ and Ni 2+ species during the pyrolysis, while the existence of Co 2+ and Ni 2+ in the hybrids indicates the surface oxidation by the atmosphere [29,37].

Electrochemical Performance of the Hybrid
The electrochemical activities of the CoxNiy/N-C hybrids were evaluated in an electrochemical cell containing 100 mL 0.1 M NaOH aqueous solution using CoxNiy/N-C hybrid-modified SPEs.For comparison, bare SPE, Co/N-C hybrid-modified SPE, and Ni/N-C hybrid-modified SPE were also measured under the same conditions.Figure 3a   We further investigated the performance of hybrid-modified SPEs in the presence of 1 mM glucose.As shown in Figure 3b, after the addition of 1 mM glucose, the oxidation current of the Co 1 Ni 1 /N-C hybrid-modified SPE shows an increase starting from around 0.2 V, while the current of the bare SPE hardly changes, indicating high electrocatalytic activity toward the glucose of the hybrid.An obvious increase in the oxidation current was also observed for other types of hybrid-modified SPEs in the presence of 1 mM glucose (Figure S4).Additionally, the Co x Ni y /N-C hybrid starts to show the current increment at around 0.2 V, which is lower than that of the pristine Co/N-C (~0.4 V) and Ni/N-C (~0.5 V) hybrids (Figure S4).The lower onset oxidation potential of the Co x Ni y /N-C hybrid indicates the synergistic effect between the Co and Ni species for the easier electro-oxidation of glucose.The sensing mechanism can be explained as follows [15], In the alkaline solution, high-valence metallic species, Ni (III) and Co (IV), are responsible for the oxidation of glucose to gluconolactone (Figure 3c).Meanwhile, the synergetic effect between the Co and Ni species, along with the porous N-doped carbon network, provides large active sites and promotes the electron transfer process, thereby promoting the electro-oxidation of glucose.
We also found that the hybrid-modified SPEs showed the largest current increment at around 0.6 V in the presence of 1 mM glucose.At the oxidation potential of 0.6 V, the Co 1 Ni 1 /N-C hybrid-modified SPE exhibits the largest current increment, further confirming the optimal Co/Ni ratio of 1:1 for glucose sensing (Figure S5).Therefore, the Co 1 Ni 1 /N-C hybrid-modified SPE was employed as the optimal electrode for the subsequent studies of glucose detection.

Glucose Detection with the Co 1 Ni 1 /N-C Hybrid-Modified SPE
Figure 4a shows the CV curves of the Co 1 Ni 1 /N-C hybrid-modified SPEs in the presence of 1 mM glucose at different scan rates ranging from 2.5 mV/s to 150 mV/s.Both anodic and cathodic currents increase with the increase in the scan rate in the presence of 1 mM glucose.The anodic current at 0.6 V linearly increases with the square root of the scan rate, suggesting that the glucose oxidation is a diffusion-controlled process (inset in Figure 4a) [38].Figure 4b indicates that the oxidation current of the CO 1 Ni 1 /N-C hybrid-modified SPE increases with the increase in the glucose concentration from 0 mM to 10 mM.We further plotted the oxidation current of the CO 1 Ni 1 /N-C hybrid-modified SPE as a function of glucose concentration at different applied potentials (Figure S6a).The sensitivity of the CO 1 Ni 1 /N-C hybrid-modified SPE for glucose oxidation reaches the maximum at the applied potential of 0.6 V (Figure S6b).Therefore, the applied potential of 0.6 V was employed to perform i-t measurement of the CO 1 Ni 1 /N-C hybrid-modified SPE for glucose in a 0.1 M NaOH aqueous solution.The amperometric response of Co1Ni1/N-C hybrid-modified SPEs was recorded by successive addition of various concentrations of glucose into a 0.1 M NaOH aqueous solution under magnetic stirring conditions (Figure 5a).A steady current can be achieved in less than 5 s after each addition of glucose.We find that the response current at 50 s of the Co1Ni1/N-C hybrid-modified SPEs to glucose shows two linear ranges (Figure 5b).The low linear range is from 0.5 µM to 1 mM (R 2 = 0.987) with a sensitivity of 1041.7 µA•mM −1 •cm −2 (inset in Figure 5b).The linear range in the high concentration region is from 1 mM to 10 mM (R 2 = 0.980) with a sensitivity of 75.7 µA•mM −1 •cm −2 .The detection limit was estimated to be 0.11 µM (S/N = 3), which is lower than most similar non-enzymatic glucose sensors reported (Table S3).The amperometric response of Co 1 Ni 1 /N-C hybrid-modified SPEs was recorded by successive addition of various concentrations of glucose into a 0.1 M NaOH aqueous solution under magnetic stirring conditions (Figure 5a).A steady current can be achieved in less than 5 s after each addition of glucose.We find that the response current at 50 s of the Co 1 Ni 1 /N-C hybrid-modified SPEs to glucose shows two linear ranges (Figure 5b).The low linear range is from 0.5 µM to 1 mM (R 2 = 0.987) with a sensitivity of 1041.7 µA•mM −1 •cm −2 (inset in Figure 5b).The linear range in the high concentration region is from 1 mM to 10 mM (R 2 = 0.980) with a sensitivity of 75.7 µA•mM −1 •cm −2 .The detection limit was estimated to be 0.11 µM (S/N = 3), which is lower than most similar non-enzymatic glucose sensors reported (Table S3).
glucose at a scan rate of 50 mV•s −1 .
The amperometric response of Co1Ni1/N-C hybrid-modified SPEs was recorded by successive addition of various concentrations of glucose into a 0.1 M NaOH aqueous solution under magnetic stirring conditions (Figure 5a).A steady current can be achieved in less than 5 s after each addition of glucose.We find that the response current at 50 s of the Co1Ni1/N-C hybrid-modified SPEs to glucose shows two linear ranges (Figure 5b).The low linear range is from 0.5 µM to 1 mM (R 2 = 0.987) with a sensitivity of 1041.7 µA•mM −1 •cm −2 (inset in Figure 5b).The linear range in the high concentration region is from 1 mM to 10 mM (R 2 = 0.980) with a sensitivity of 75.7 µA•mM −1 •cm −2 .The detection limit was estimated to be 0.11 µM (S/N = 3), which is lower than most similar non-enzymatic glucose sensors reported (Table S3).Lac, Fru, AA, UA, DA, ATP, and NaCl, which coexist with glucose in biological fluids, are major interference species for the detection of glucose.We studied the current response of the Co1Ni1/N-C hybrid-modified SPE after the successive addition of 1 mM glucose and interferences of 2 mM Lac, 2 mM Fru, 2 mM AA, 2 mM UA, 2 mM DA, 2 mM ATP, and 30 mM NaCl in the 0.1 M NaOH aqueous solution (Figure 5c).However, we found that compared with the current change induced by 1 mM glucose, the Co1Ni1/N-C hybrid-modified SPE exhibits a negligible current change after the addition of interferences, indicating the good selectivity of the Co1Ni1/N-C hybrid-modified SPE.
The reproducibility of the CO1Ni1/N-C hybrid-modified SPE was evaluated using five independent electrodes for the detection of 1 mM glucose in a 0.1 M NaOH aqueous solution (Figure 6a).The relative standard deviation (RSD) of the five current responses is 4.84%, indicating the good reproducibility of the electrode.The stability experiment was conducted by measuring the current response of the prepared CO1Ni1/N-C hybrid-

Glucose Detection Using Real Samples
To assess the feasibility of the Co1Ni1/N-C hybrid-modified SPE, human serum samples were collected from volunteers in Chongqing University Hospital to evaluate the glucose sensing performance.The samples were used for the experiment with the consent of the volunteers and Chongqing University Hospital.For analysis, 6 µL serum was first diluted using 24 µL 0.1 M NaOH aqueous solution and then dropped on the SPE.Amperometry measurement (i-t) was performed at the applied potential of 0.6 V for 60 s to deter-

Glucose Detection Using Real Samples
To assess the feasibility of the Co 1 Ni 1 /N-C hybrid-modified SPE, human serum samples were collected from volunteers in Chongqing University Hospital to evaluate the glucose sensing performance.The samples were used for the experiment with the consent of the volunteers and Chongqing University Hospital.For analysis, 6 µL serum was first diluted using 24 µL 0.1 M NaOH aqueous solution and then dropped on the SPE.Amperometry measurement (i-t) was performed at the applied potential of 0.6 V for 60 s to determine the glucose concentration in the diluted serum samples (Figure S7).The current response at 50 s was extracted to calculate the corresponding glucose concentration based on the calibration curve.For comparison, a blood glucose meter and its electrode (accuracy: ≤±20%, precision: CV < 7.5%, Yuwell 660, Zhenjiang, China) were employed to determine the glucose levels in the serum samples.As shown in Table 1, compared with the glucose concentration determined by the glucose meter, the Co 1 Ni 1 /N-C hybrid-modified SPE shows similar results with a relative standard deviation (RSD) of less than 3.01%.These results indicate that the Co 1 Ni 1 /N-C hybrid-modified SPE demonstrates good feasibility for detecting glucose in real samples.

Conclusions
In summary, Co x Ni y /N-C hybrids with different Co/Ni ratios were synthesized through the carbonization of MOF precursors.The obtained hybrids exhibited good electrocatalytic activities toward glucose.We found that the microstructure and electrocatalytic performance of the hybrid are dependent on the Co/Ni ratio.The hybrid-modified SPE with the optimal Co/Ni ratio of 1:1 for the electro-oxidation of glucose shows two linear detection ranges (0.5 µM to 1 mM, 1 mM to 10 mM) with a sensitivity of 1041.7 µA•mM −1 •cm −2 and 75.7 µA•mM −1 •cm −2 , respectively.The detection limit of the Co 1 Ni 1 /N-C hybridmodified SPE for glucose was calculated as being as low as 0.11 µM.The high-sensing performance of the Co 1 Ni 1 /N-C hybrid can be ascribed to the synergistic effect between the Co and Ni species, along with the porous N-dopant carbon matrix.The feasibility of the Co 1 Ni 1 /N-C hybrid-modified SPE for glucose detection in real serum samples was also validated, indicating a promising candidate for the non-enzymatic detection of glucose.
C for 2 h under N 2 gas to achieve the Co x Ni y /N-C hybrids with different Co/Ni ratios, labeled as Co 3 Ni 1 /N-C, Co 1 Ni 1 /N-C, and Co 1 Ni 3 /N-C, respectively, according to their feeding ratios.For comparison, Co/N-C and Ni/N-C hybrids were synthesized using the same protocol except for the addition of 6.4 mM of Co(NO 3 ) 2 •6H 2 O and 6.4 mM Ni(NO 3 ) 2 •6H 2 O, respectively, into the Zn(NO 3 ) 2 •6H 2 O aqueous solution to prepare the metal salt solution instead of the Co(NO 3 ) 2 •6H 2 O and Ni(NO 3 ) 2 •6H 2 O mixture.
), (200), and (220) planes of the face-centered cubic (FCC) structures of Co (JCPDS No. 04-0850) and Ni (JCPDS No. 04-0850), respectively [28].No other obvious peaks are observed, suggesting the evaporation of the Zn species during the pyrolysis process.Figure 2b shows the zoomed-in (111) peak of the hybrids.Compared with pristine Co/N-C, the zoomed-in (111) peak of the CoxNiy/N-C hybrid gradually shifts towards a higher 2θ value with the increase in the Ni amount.This is due to the la ice contraction of the Ni alloying with Co, confirming the formation of the Co-Ni alloy [20,29].No other planes were observed for the CoxNiy/N-C hybrids, implying that the formation of the Co-Ni alloy has not destroyed the crystal structure of Co or Ni due to the similar la ice parameters of Co and Ni.

Figure 2 .
Figure 2. XRD pa erns (a) and the corresponding zoomed-in XRD pa erns (b) of the hybrids.XPS spectra of C 1s (c), N 1s (d), Co 2p (e), and Ni 2p (f) for the Co/N-C, Ni/N-C, and Co1Ni1/N-C hybrids.The chemical compositions and valence states of the elements in the hybrids were characterized using XPS (Figures 2c-f and S3).Three peaks in the high-resolution C 1s spectra located at 284.8 eV, 285.6 eV, and 289.1 eV, are assigned to C=C, C-N and O-C-O,
shows the zoomed-in (111) peak of the hybrids.Compared with pristine Co/N-C, the zoomed-in (111) peak of the CoxNiy/N-C hybrid gradually shifts towards a higher 2θ value with the increase in the Ni amount.This is due to the la ice contraction of the Ni alloying with Co, confirming the formation of the Co-Ni alloy [20,29].No other planes were observed for the CoxNiy/N-C hybrids, implying that the formation of the Co-Ni alloy has not destroyed the crystal structure of Co or Ni due to the similar la ice parameters of Co and Ni.

Figure 2 .
Figure 2. XRD pa erns (a) and the corresponding zoomed-in XRD pa erns (b) of the hybrids.XPS spectra of C 1s (c), N 1s (d), Co 2p (e), and Ni 2p (f) for the Co/N-C, Ni/N-C, and Co1Ni1/N-C hybrids.The chemical compositions and valence states of the elements in the hybrids were characterized using XPS (Figures2c-f and S3).Three peaks in the high-resolution C 1s spectra located at 284.8 eV, 285.6 eV, and 289.1 eV, are assigned to C=C, C-N and O-C-O,
. The amount of pyridinic and pyrrolic N in the Co 1 Ni 1 /N-C hybrid (72.1%) is the highest among these hybrids (68.3% for Co/N-C, 69.5% for Ni/N-C, 71.4% for CO 3 Ni 1 /N-C, and 70.1% for CO 1 Ni 3 /N-C, respectively) (Table reveal that the Co 2p spectra of the Co x Ni y /N-C and Co/N-C hybrids compose the two main peaks of Co 2p 3/2 and Co 2p 1/2 , which are further deconvoluted into four subpeaks, corresponding to Co0 (~778.5 eV and ~794.1 eV) and Co 2+ (~780.4eV and ~797.0 eV), and two satellites at around 784.4 eV and 804.2 eV [15].The Ni 2p spectra of the Co x Ni y /N-C and Ni/N-C hybrids have confirmed the presence of metallic Ni at around 852.8 eV and 870.3 eV, respectively (Figures

3. 2 .
Electrochemical Performance of the Hybrid The electrochemical activities of the Co x Ni y /N-C hybrids were evaluated in an electrochemical cell containing 100 mL 0.1 M NaOH aqueous solution using Co x Ni y /N-C hybridmodified SPEs.For comparison, bare SPE, Co/N-C hybrid-modified SPE, and Ni/N-C hybrid-modified SPE were also measured under the same conditions.Figure 3a shows the CV curves of the SPEs with and without the modification of the hybrids in a 0.1 M NaOH aqueous solution at a scan rate of 50 mV•s −1 .The current of the hybrid-modified SPEs shows a huge improvement compared with that of the bare SPE.The Ni/N-C-modified SPE shows an anodic peak current at around 0.51 V, indicating the oxidation of Ni 2+ to Ni 3+ , while the Co/N-C-modified SPE exhibits an anodic peak current of Co 2+ to Co 3+ at around 0.16 V [21].Although the oxidation peaks of the CO 3 Ni 1 /N-C and CO 1 Ni 3 /N-C are located between those of the pristine Co/N-C and Ni/N-C hybrids due to the formation of the Co-Ni alloy, the peak current is smaller than that of the pristine Ni/N-C (Figure 3a).Interestingly, the Co 1 Ni 1 /N-C displays the highest oxidation current among the hybrids, indicating the significant role of the Co/Ni ratio in dictating the electrocatalytic performance of the hybrid.
shows the CV curves of the SPEs with and without the modification of the hybrids in a 0.1 M NaOH aqueous solution at a scan rate of 50 mV•s −1 .The current of the hybrid-modified SPEs shows a huge improvement compared with that of the bare SPE.The Ni/N-C-modified SPE shows an anodic peak current at around 0.51 V, indicating the oxidation of Ni 2+ to Ni 3+ , while the Co/N-C-modified SPE exhibits an anodic peak current of Co 2+ to Co 3+ at around 0.16 V [21].Although the oxidation peaks of the CO3Ni1/N-C and CO1Ni3/N-C are located between those of the pristine Co/N-C and Ni/N-C hybrids due to the formation of the Co-Ni alloy, the peak current is smaller than that of the pristine Ni/N-C (Figure 3a).Interestingly, the Co1Ni1/N-C displays the highest oxidation current among the hybrids, indicating the significant role of the Co/Ni ratio in dictating the electrocatalytic performance of the hybrid.

Figure 3 .
Figure 3. (a) CV curves of SPEs with and without the modification of the hybrid in 0.1 M NaOH aqueous solution at a scan rate of 50 mV•s −1 .(b) CV curves of the bare SPE and Co 1 Ni 1 /N-C hybrid-modified SPE in 0.1 M NaOH aqueous solution in the absence and presence of 1 mM glucose at a scan rate of 50 mV•s −1 .(c) Schematic illustration of the electro-oxidation of glucose on the Co-Ni hybrid-modified SPE.

Figure 4 .
Figure 4. CV curves of the Co1Ni1/N-C hybrid-modified SPE in aqueous solutions containing (a) 1 mM glucose at various scan rates (inset: the corresponding anodic peak current as a function of the square root of the scan rate at the applied potential of 0.6 V), and (b) different concentrations of glucose at a scan rate of 50 mV•s −1 .

Figure 4 .
Figure 4. CV curves of the Co 1 Ni 1 /N-C hybrid-modified SPE in aqueous solutions containing (a) 1 mM glucose at various scan rates (inset: the corresponding anodic peak current as a function of the square root of the scan rate at the applied potential of 0.6 V), and (b) different concentrations of glucose at a scan rate of 50 mV•s −1 .

Figure 5 .
Figure 5. Amperometric response of the Co1Ni1/N-C hybrid-modified SPE after successive addition of different concentrations of glucose into a 0.1 M NaOH aqueous solution (a) and the corresponding calibration curve for glucose detection (inset: linear fi ing of the low concentration regime) (b).(c) Amperometric response of the Co1Ni1/N-C hybrid-modified SPE after the successive addition of glucose and interferences.

Figure 5 .
Figure 5. Amperometric response of the Co 1 Ni 1 /N-C hybrid-modified SPE after successive addition of different concentrations of glucose into a 0.1 M NaOH aqueous solution (a) and the corresponding calibration curve for glucose detection (inset: linear fitting of the low concentration regime) (b).(c) Amperometric response of the Co 1 Ni 1 /N-C hybrid-modified SPE after the successive addition of glucose and interferences.Lac, Fru, AA, UA, DA, ATP, and NaCl, which coexist with glucose in biological fluids, are major interference species for the detection of glucose.We studied the current response of the Co 1 Ni 1 /N-C hybrid-modified SPE after the successive addition of 1 mM glucose and interferences of 2 mM Lac, 2 mM Fru, 2 mM AA, 2 mM UA, 2 mM DA, 2 mM ATP, and 30 mM NaCl in the 0.1 M NaOH aqueous solution (Figure 5c).However, we found that compared with the current change induced by 1 mM glucose, the Co 1 Ni 1 /N-C hybrid-modified SPE exhibits a negligible current change after the addition of interferences, indicating the good selectivity of the Co 1 Ni 1 /N-C hybrid-modified SPE.The reproducibility of the CO 1 Ni 1 /N-C hybrid-modified SPE was evaluated using five independent electrodes for the detection of 1 mM glucose in a 0.1 M NaOH aqueous solution (Figure 6a).The relative standard deviation (RSD) of the five current responses is 4.84%, indicating the good reproducibility of the electrode.The stability experiment was conducted by measuring the current response of the prepared CO 1 Ni 1 /N-C hybridmodified SPE for 1 mM glucose for 7 days.As shown in Figure 6b, the CO 1 Ni 1 /N-C hybrid-modified SPE retained 90.22% of the initial responses for 1 mM glucose after 7 days, suggesting good stability.Chemosensors 2024, 12, x FOR PEER REVIEW 8 of 11

Figure 6 .
Figure 6.The current response of the CO1Ni1/N-C hybrid-modified SPE (a) was evaluated using five independent electrodes and (b) was evaluated at different days for the detection of 1 mM glucose in a 0.1 M NaOH aqueous solution.

Figure 6 .
Figure 6.The current response of the CO 1 Ni 1 /N-C hybrid-modified SPE (a) was evaluated using five independent electrodes and (b) was evaluated at different days for the detection of 1 mM glucose in a 0.1 M NaOH aqueous solution.
Figure S1: N2 adsorption/desorption isotherm of Co 1 Ni 1 /N-C hybrid; Table S1: Specific surface areas of hybrids calculated by BET method; Figure S2: The TEM images and the corresponding elemental mapping images of (a) Co 3 Ni 1 /N-C and (b) Co 1 Ni 3 /N-C hybrids for C, N, O, Co, and Ni.The scale bar is 500 nm; Figure S3: XPS spectra of C 1s, N 1s, Co 2p, and Ni 2p for (a) Co 3 Ni 1 /N-C and (b) Co 1 Ni 3 /N-C hybrids; Table S2: N contents calculated from XPS spectra; Figure S4: CV curves of SPEs modified with (a) Co/N-C, (b) Ni/N-C, (c) Co 1 Ni 3 /N-C, and (d) Co 3 Ni 1 /N-C hybrids in 0.1 M NaOH aqueous solution in the absence and presence of 1 mM glucose at a scan rate of 50 mV•s −1 ; Figure S5: The current increment extracted from the CVs of hybrid-modified SPEs with different Co/Ni ratios at 0.6 V in presence of 1 mM glucose at a scan rate of 50 mV•s −1 ; Figure S6: Current response of CO 1 Ni 1 /N-C-modified SPE as a function of glucose concentration in 0.1 NaOH aqueous solution at various applied potentials (a) and the corresponding sensitivity at different applied potentials (b); Table S3: Comparison of the sensing performance of Co 1 Ni 1 /N-C hybrid-modified SPE prepared in this work with other non-enzymatic

Table 1 .
Glucose detection in human serum samples using a blood glucose meter and the Co 1 Ni 1 /N-C hybrid-modified SPE.