An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-Nitroquinoline N-Oxide in Biologic Matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study

Various factors leads to cancer; among them oxidative damage is believed to play an important role. Moreover, it is important to identify a method to detect the oxidative damage. Recently, electrochemical sensors have been considered as the one of the most important techniques to detect DNA damage, owing to its rapid detection. However, electrode materials play an important role in the properties of electrochemical sensor. Currently, researchers have aimed to develop novel electrode materials for low-level detection of biomarkers. Herein, we report the facile hydrothermal synthesis of NiCo2O4 micro flowers (MFs) and NiCo2S4 micro spheres (Ms) and evaluate their electrochemical properties for the detection of carcinogen-causing biomarker 4-nitroquinoline n-oxide (4-NQO) in human blood serum and saliva samples. Moreover, as-prepared composites were fabricated on a glass carbon electrode (GCE), and its electrochemical activities for the determination of 4-NQO were investigated by using various electrochemical techniques. Fascinatingly, the NiCo2S4-Ms showed a very low detection limit of 2.29 nM and a wider range of 0.005 to 596.64 µM for detecting 4-NQO. Finally, the practical applicability of NiCo2S4-Ms in the 4-NQO spiked human blood serum and saliva samples were also investigated.


Introduction
Currently, bimetallic oxide and sulfide-based nanoparticles have been widely used in the field of electrochemical application [1][2][3]. A well-known electrode material with superior electrocatalytic property prepared based on nickel and cobalt due its higher surface area, structures and unique morphologies than monometallic oxides or sulfides [4][5][6][7][8]. Thus, we have synthesized hierarchical spinel NiCo 2 O 4 -MFs and NiCo 2 S 4 -Ms as an electrode material for the electrocatalytic reduction of 4-NQO detection. The better response occurs on the NiCo 2 S 4 -Ms modified electrode because sulfides provide the lower bandgap energy and less electronegativity than oxides [8][9][10]. The synthesis

Microscopic and Elemental Analysis of Synthesized Nanomaterials
The morphologic structure of synthesis nanoparticles micro flowers and microspheres were obtained using FESEM. Figure 1A characteristic the morphology of NiCo 2 O 4 , which appears as a flower-like structure and Figure 1B, shows the magnified microscopic image of NiCo 2 O 4 in which clearly demonstrated that the numerous nano-needles are shrinks together and forms a micro spiky structure. Moreover, the FESEM images of the NiCo 2 S 4 Figure 1C,D show that obtained particles were micro structured spheres, which were regularly distributed with an average particle size of 1.64 µm. Figure 2A-D illustrates the elemental mapping of NiCo 2 S 4 Ms (A), which confirmed the presence of Ni (B), Co (C) and S (D) elements. In addition, the EDX analysis were taken to investigate the elemental composition present in NiCo 2 S 4 Ms. Figure 2E shows the EDX spectrum of NiCo 2 S 4 Ms with the expected signal response of the elements Ni, Co and S. The inset image of Figure 2E

XRD and XPS Analysis of NiCo 2 S 4 -Ms and NiCo 2 O 4 -MFs
The XRD patterns of the as synthesized NiCo 2 S 4 -Ms and NiCo 2 O 4 -MFs are shown in Figure 3A. The XRD pattern of NiCo 2 S 4 -Ms Figure 3Aa shows the characteristic peaks at 16 [25]. Moreover, Figure 3Ab shows the signal response at 18

EIS and Electrochemical Investigation of Different Electrodes
Electrochemical impedance spectroscopy (EIS) is a method to investigate the interfacial effects between electrolyte and surface of the electrode. Figure Figure 4A, where R ct denotes the charge transfer resistance, Z W , R s and C dl refer to Warburg impedance, ohmic resistance and the double layer electron-transfer resistance, respectively. Moreover, the R ct value of bare GCE, NiCo 2 O 4 -MFs/GCE and NiCo 2 S 4 -Ms/GCE were measured to be 250.7 Ω, 214.2 Ω and 64.25 Ω, respectively. As can be seen that the lowest R ct value were obtained for NiCo 2 S 4 -Ms electrode. This was due to the microsphere structure possessing the larger surface area; when it contacted the electrolyte, it may be more effective and efficient in capturing the active materials.  Figure 4B as can be seen that, the highest reduction and reduction peak current and lower peak-to-peak separation (∆Ep) value of 89.24 mV were observed for the NiCo 2 S 4 -Ms/GCE. Moreover, the peak-to-peak separation (∆Ep) of unmodified electrode (a) and NiCo 2 O 4 -MFs/GCE (b) were measured to be 189.51 and 145.36 mV, respectively. Figure 4C shows the different scan rates at NiCo 2 S 4 -Ms/GCE, in which the redox peak current increased consistently and their linear relationship with the square root of scan rates were plotted as shown in Figure 4D. Furthermore, the electrochemical active surface area value was measured based on the Randel's Sevcik equation (I). The electrochemical active surface area of bare GCE (a), NiCo 2 O 4 -MFs/GCE (b) and NiCo 2 S 4 -Ms/GCE were calculated to be 0.082, 0.102 and 0.161 cm 2, respectively. The results indicate the combination of bimetallic sulfides increased the surface area and showed excellent electrochemical contact between the surface of electrode and electrolyte solution. Ip = 2.69 × 10 5 n 3/2 AD 1/2 Cv 1/2 (1)

Electrochemical Activity and Different pH at NiCo 2 S 4 -Ms/GCE
As shown in Figure 5A, the CV's with different electrodes, bare GCE (a), NiCo 2 O 4 -MFs/GCE (b) and NiCo 2 S 4 -Ms/GCE (c) were recorded in 0.1 M of phosphate buffer solution (PB) pH 7 containing 100-µM 4-NQO with the fixed scan rate of 0.05 V/s. The lower reduction peak current exhibited an unmodified GCE, which suggest the poor conductivity on the electrode surface. Furthermore, when the electrode was modified with NiCo 2 O 4 -MFs/GCE there was a drastically increase in the current. The excellent peak current of −26 µA with the minimized reduction potential of −0.29 V was noted on NiCo 2 S 4 -Ms/GCE, which suggest that the larger surface area of NiCo 2 S 4 -Ms can considerably enhance the sensitivity of the electrode. Moreover, the probable electrochemical reduction mechanism of 4-NQO on NiCo 2 S 4 -Ms/GCE are shown in Figure 6. Typically, 4-nitroquinoline n-oxide is irreversibly reduced to form 4-hydroxyaminoquinoline n-oxide. After this, the oxidation of 4-hydroxyaminoquinoline n-oxide occurs, which is subsequently reduced to 4-nitrosoquinoline n-oxide at the increasing scan. The accumulation time is one of the important parameters in electrochemical sensors to improve the sensitivity. Therefore, the accumulation time of the proposed sensor was studied in 0.1-M pH 7 containing 100-µM 4-NQO using the CV technique and the corresponding current response versus time were plotted as shown in Figure S2. The obtained results indicate that the reduction peak increased with the increasing accumulation time. However, the accumulation time exceeds 20 s, the reduction peak current of 4-NQO decreased. This was due to the adsorption taken at the surface of the electrode, which may have resisted the active site of the NiCo 2 S 4 -Ms and the surface reached its saturation point at 20 s. Thus, the optimal accumulation time of 20 s was preferred to achieve the high sensitivity for the proposed sensor.  Furthermore, the influence of pH at NiCo 2 S 4 -Ms/GCE for the detection of 4-NQO was investigated by varying the PB pH from 3 to 11 at a fixed scan rate of 0.05 Vs −1 . Figure 5B displays the CV's curves of different pH 3 to 11 containing 100-µM 4-NQO. The well-shaped peak with higher reduction peak current of −5.29 µA was noted at pH 7 Figure 5C. At some point, the peak current started to decrease, which indicates that the biomolecules reached its maximum pH value of 9-11. Hence, the pH 7 was chosen as the enhanced pH for the reduction of 4-NQO. In addition, the plot between the reduction peak potential and pH were plotted as shown in Figure 5D, which was linear, and its regression equation were represented as E p (V) = −0.056 pH −0.624. The obtained slope value of −56 mV/pH indicates that the reaction was equal number of proton and electron transferred.

Influence of Different Concentration and Scan Rate
In addition, the influence of various concentration of 4-NQO at as-prepared NiCo 2 S 4 -Ms/GCE were analyzed in 0.1-M pH 7 at fixed scan window of 0.05 Vs −1 . As shown in Figure 7A, the CV curve were recorded for the increasing concentration of 4-NQO. For every sequential addition of 4-NQO from 25 to 250 µM the reduction peak current also increased linearly. Finally, the linear relationship of reduction peak current versus the concentration of 4-NQO were plotted as Figure 7B. Moreover, the regression equation is written to be y = 0.0253x + 2.2034 with the correlation coefficient of R 2 = 0.9938. The obtained results confirm that, the as-prepared NiCo 2 S 4 -Ms is a promising electrode material for the rapid detection of 4-NQO without fouling. In order to study the effects of scan rates at NiCo 2 S 4 -Ms/GCE was investigated by CV techniques. Figure 7C, indicates the CVs of different scant rates from 20 to 300 mVs −1 in 0.1-M PB pH 7 contains 100-µM 4-NQO.With consistently increasing scan rates, there was a linear increase in the cathodic peak current. Figure 7D shows the linear relationship between the cathodic peak current of 4-NQO versus square root of the scan rates. In addition, the regression equation was calculated to be y = 8.3872x − 0.0241 with the correlation coefficient of R 2 = 0.9908, which indicates that reduction of 4-NQO at NiCo 2 S 4 -Ms/GCE was a diffusion-controlled process.

DPV Analysis of 4-NQO Ions at NiCo 2 S 4 -Ms/GCE Techniques
Differential pulse voltammetry common techniques were used to measure the essential electrochemical parameters such as linear range, sensitivity and limit of detection (LOD). Hence, the DPV technique was used to detect the 4-NQO at NiCo 2 S 4 -Ms/GCE in 0.1-M pH 7 at a fixed potential of 0.4 to −0.6 V. Figure 8A displays the DPV curve of increasing concentration of 4-NQO. The obtained results indicate the reduction peak current increased with increasing concentration of 4-NQO. The NiCo 2 S 4 -Ms/GCE modified electrode showed an excellent linear relationship between the reduction peak current and the concentration of 4-NQO, which were plotted as shown in Figure 8B. Moreover, the linear regression equation was calculated to be ip a (µA) = 0.4014 µM -0.0153 (R 2 = 0.9985). Wherein, the NiCo 2 S 4 -Ms/GCE shows the wider range of 0.005 to 596.64-µM 4-NQO. In addition, the LOD of the as-prepared sensor was measured using the standard formula of LOD = 3σ/S, where the σ and S, is the standard deviation and slope of the curve, respectively. Moreover, the LOD of NiCo 2 S 4 -Ms/GCE for the detection of 4-NQO were measured to be 2.29 nM. Moreover, the proposed sensor wes compared with previously reported 4-NQO sensor as the results as-prepared NiCo 2 S 4 -Ms/GCE shows the very low detection limit Table 1. Thus, it is a promising electrode material for the detection of 4-NQO.

Interference and Stability Studies of NiCo 2 S 4 -Ms/GCE
Selectivity and stability are the most considered parameter in electrochemical studies. Hence, the selectivity of the sensor was examined using NiCo 2 S 4 -Ms/GCE with 4-NQO, as well as in the presence of possible bioactive and nitro compounds. The selectivity of the prepared sensor was studied using DPV techniques. The DPV results of the NiCo 2 S 4 -Ms/GCE in presence of 0.5 µM 4-NQO and 0.7 µM possible interferents such as 3-nitro-l-tryosine (3-NT), 4-nitrophenol (4-NP), chloramphenicol (CAP), nitro benzene (NB) and continued addition of 1.5 µM common biologic analytes glucose (Glu), ascorbic acid (AA), hydrogen peroxide (H 2 O 2 ) and dopamine (DA) are shown in Figure S3. The related nitro compounds such as 3-NT, 4-NP, NB, CAP showed minor interference with the 4-NQO due to structural similarity. However, the corresponding relative error was found less than 7% as shown in Figure 9A. Notably, most of the other nitro compounds show their reduction peaks at high overpotential of −0.50 to −0.60 V, which is far from the reduction potential of 4-NQO. In other words, although all these compounds have nitro groups, they require different energy to be reduced, which provides the electrode a good selectivity. Approximately three-fold excess concentrations of Glu, AA, H 2 O 2 and DA did not show any significant interference, indicating that the method is selective in biologic samples. Moreover, the working stability of NiCo 2 S 4 -Ms/GCE were examined using the DPV technique. Figure 9B shows the stability current response of the prepared sensor in 0.1 M pH 7 containing 10-µM 4-NQO. After 15 days usage, the sensor maintained a stability of 94.59%. Thus, the stability test of the NiCo 2 S 4 -Ms/GCE confirms the outstanding working stability for the detection of 4-NQO.

Real Sample Analysis
The practical applicability of prepared sensor was examined in the biologic sample human blood serum and saliva samples. The preparation of biologic samples was diluted with buffer solution and the known concentration of 4-NQO were spiked. Finally, DPV techniques were carried out for the prepared real samples. Fascinatingly, the blood serum and saliva samples were showed outstanding found and recovery rates, which are tabulated in Table 2. At the end, the prepared NiCo 2 S 4 -Ms/GCE established as the effective electrode for the real time applicability.

Synthesis Method for NiCo 2 S 4 -Ms and NiCo 2 O 4 -MFs
All chemicals were purchased from Sigma-Aldrich in analytical grade and used without any further purification. The details of chemical purchases, preparation methods of buffer solutions and instrumentation techniques are detailed in the supplementary data (S1). The NiCo 2 O 4 -MFs and NiC 2 S 4 -Ms were synthesized by the hydrothermal method. Briefly, a 1:2 molar ratio of Ni(CH 3 COO) 2 ·4H 2 O and Co(CH 3 COO) 2 ·4H 2 O were thoroughly dissolved in 50 mL of C 2 H 6 O 2 under magnetic stirring for 30 min. Then the mixture was carefully transferred into a 100 mL Teflon-lined stainless-steel autoclave and maintained for 180 • C for 12 h in a hydrothermal oven. After 12 h, the reaction mixture was allowed to cool room temperature and centrifuged with water and ethanol to collect the NiCo 2 O 4 -MFs precipitate. Finally, the collected precipitate was dried in air at 80 • C for 10 h [30]. To synthesis, the NiCo 2 S 4 -Ms, a 1:2 molar ratio of the NiSO 4 ·6H 2 O and CoSO 4 ·7H 2 O were dissolved in 0.5 M Na 2 S·5H 2 O. Then the same procedure was followed to obtain the NiCo 2 S 4 -Ms (Scheme 1). Scheme 1. Synthesis methodology of NiCo 2 S 4 -Ms and its electrocatalytic properties for the detection of 4-nitroquinoline n-oxide.

Fabrication of NiCo 2 S 4 -Ms Modified GCE
The surface of GCE was cleaned by 0.5 mg of alum in a slurry. Then the GCE was dipped into an ethanol solution and sonicated for 10 min. After this, the GCE was pre-cleaned by cycling between −0.8 to 0.8 V in PB pH 7 for the 25 continuous cycle. Then, 1 mg of as-prepared NiCo 2 S 4 -Ms was dispersed in 1 mL of ethanol and sonicated for 20 min. Approximately 6 µL of NiCo 2 S 4 -Ms suspension was drop-cast on the surface of GCE and dried in a 50 • C oven. Finally, the as-prepared NiCo 2 S 4 -Ms modified GCE was used for the electrochemical characterization.

Materials and Reagents
Nickel acetate Ni(CH 3 COO) 2 ·4H 2 O, cobalt acetate Co(CH 3 COO) 2 ·4H 2 O, ammonium hydroxide (NH 4 OH), nickel sulfate NiSO 4 ·6H 2 O, cobalt sulfate CoSO 4 ·7H 2 O, 4-nitroquinoline n-oxide, uric acid (UA), ascorbic acid (AA), dopamine (DA), glucose (GLU), 3-nitrotyrosine (3-NT), L-cysteine and H 2 O 2 and all other chemicals were purchased from Sigma-Aldrich and used as received. Double-distilled water was used for all the experiments. 0.1-M phosphate buffer (PB) (pH 7.0) prepared from sodium dihydrogen phosphate and disodium hydrogen phosphate was used as supporting electrolyte. Human blood serum and saliva samples were acquired from Chang Gung Medical Hospital, Taoyuan, Taiwan. The research protocols of human blood serum and saliva samples experiments were followed as per the laws and institutional guidelines of Chang Gung Medical Hospital (CGMH), Taiwan.

Methods
The surface modification of the as-formed composite was examined using field emission scanning electron microscope (FESEM-JEOL-7600F, Jeol instruments, Musashino, Akishima, Tokyo, Japan): Ingredients of the elemental composition and elemental mapping were analyzed by energy-dispersive X-ray spectroscopy (EDX) with HORIBA EMAX X-ACT (Horiba instruments, AkzoNobel House, Singapore). The quantitative analysis and defects and disordered nature of the as-prepared composite were investigated by PerkinElmer PHI-5702 (PerkinElmer Inc., Waltham, MA, USA).
The crystalline nature of the composite was examined by XRD, XPERT-PRO spectrometer (Malvern Panalytical B.V., Almelose Aa, Netherland). The electrochemical properties and electrocatalytic activity were examined using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV); i-t amperometry was carried out CHI 1205A (CH instruments, Inc. Austin, TX, USA). The CHI instrument consists of a three-electrode system, in which the platinum wire and Ag/AgCl (sat. KCl) were used as auxiliary and reference electrodes, and a pre-washed GCE (glassy carbon electrode) acted as a working electrode.

Conclusions
In summary, we successfully synthesized NiCo 2 S 4 -Ms and NiCo 2 O 4 -MFs through the hydrothermal method. The formation of NiCo 2 S 4 -Ms and NiCo 2 O 4 -MFs were conformed through the several analytical techniques such as FESEM, EDX, XPS and XRD. Further, the as-prepared nanocomposites were used as electrode materials for the detection of 4-NQO. Fascinatingly, the NiCo 2 S 4 -Ms modified electrode showed excellent electrochemical performances such as a wider range, low limit detection, higher selectivity and outstanding stability for the detection of 4-NQO. In addition, the practical applicability of the as-prepared nanocomposite were scrutinized in the human blood serum and saliva samples, which showed good recovery for both samples. To our knowledge, the hydrothermally synthesized NiCo 2 S 4 -Ms is the one of the most effective electrocatalysts for the detection of 4-NQO.