Highly dispersed silver imbedded into TiN submicrospheres for electrochemical detecting of hydrogen peroxide

We report the fabrication of silver nanoparticles evenly imbedded into TiN submicrospheres via one-pot solvothermal reaction and subsequent nitridation for electrochemical detecting of hydrogen peroxide. The precursor of TiO2 submicrospheres and high dispersion of silver nanoparticles are regulated by the alcoholysis of tetrabutyl titanate and reducibility of enol in vitamin C. The ion nitriding promoted the conductivity and micro-nano porous structure on the surface of TiN submicrospheres, which increase the dispersity of silver nanoparticles and make contributions to avoid aggregations. More importantly, the electrochemical response of Ag-TiN submicrospheres to H2O2 was remarkably enhanced due to the co-effects of Ag and N-doping. It provides a superior sensing performance for electrochemical detection of hydrogen peroxide at − 0.3 V with a high sensitivity of 33.25 μA mmol L−1 cm−2, wide linear range of 0.05–2100 μM and low detection limit of 7.7 nM. The fabricated sensor also reliably applied in detection of H2O2 in milk samples with good reproducibility, repeatability and storage stability.

Many methods have been proposed for the preparation of highly dispersed silver, such as spray pyrolysis 17 , electrolysis 18 , microwave plasma 19 , and chemical liquid-phase reduction process 20 . Most of the reported literatures focused on controlling the size of silver, while little on the dispersibility and few on dispersive mechanism. Immobilization of silver on organic or inorganic scaffolds has been proved to be an effective strategy against agglomeration with the improved stability [21][22][23] . Nanostructured titanium oxides as important multi-valent compound, including TiO, Ti 2 O 3 and TiO 2 , can be employed as the scaffold for silver due to their unique electrical property, non-toxic, and chemical stability. Furthermore, titanium oxides have the characteristic of controllability in micro-scale construction, which provides an efficient way to load silver. However, changes in the crystalline phase, shape and conductivity of microstructure could have led to reliability at load that caused in the availability of carrier materials for sensors [24][25][26] . It has been reported that doping with nitrogen could enhance the interior conductivity of TiO 2 due to the uniform distribution of dopants throughout the particles. As successful examples, a sugar-apple-like N-doped TiO 2 @ carbon core-shell spheres as high-rate and long-life anode materials has been synthesized by carburizing and nitriding, while the conductive N-doped carbon shell with slit pores uniformly coated on TiO 2 spheres surface revealing superior long-term cycling stability for lithium ion batteries 27,28 . Furthermore, titanium nitride (TiN) has attracted extensive interests as carrier materials because of super high electrical conductivity, biocompatibility and chemical stability [29][30][31] . Biocompatible TiN nanorod arrays fabricated by solvent-thermal synthesis and subsequent nitridation in ammonia atmosphere deliver the superior electrocatalytic activity and highly selective sensing H 2 O 2 owing to their good electronic conductivity and large surface area 32 . Robust TiN nanotubes supported Pt catalyst with enhanced catalytic activity and durability for methanol oxidation reaction exhibit small size, good dispersion and fast electron transfer due to the strong metal-support interactions of TiN nanotubes 33 . Recent researches show TiN can be employed in a wide range of applications such as biosensing 32 , pH-sensitive material 34 , electroanalysis 35 , supercapacitors 36 , and energy storage 37 . Therefore, TiN can better be employed as the scaffold for silver because of their high chemical and physical stability, environmentally non-toxic and unique electrical property. Furthermore, the electrocatalytic ability of TiN might promote the synergistic effects of silver highly dispersed nanocomposites for H 2 O 2 sensors.
In the present work, nonstoichiometric single phase TiN was employed as scaffold and solvothermal pathways in harmoy of silver dispersion and subsequent nitridation using ammonia annealing. In the solvothermal process, vitamin C in ethanol was used as a reducing agent to get the silver evenly distributed in TiN submicrospheres. Figure 1 displays the synthesis procedure for fabrication of Ag-TiN submicrospheres. Benefiting from its inexpensive, simple synthetic route and extraordinary properties for H 2 O 2 detection, this novel material is a hopeful candidate in the development of efficient nonenzymatic H 2 O 2 sensor.
One-step hydrothermal process of Ag-TiO 2 submicrospheres (Ag-TiO 2 /SMS) 38 . In the paper, vitamin C (30 mmol) and AgNO 3 (30 mg) were added to absolute ethanol (70 mL) with magnetic stirring, and then adding TBOT (8 mmol) to solution drop by drop form clear to brown color (Ag / Ti source (m %) is 10%). Subsequently, the mixture was transferred into 100 mL Teflon-line stainless autoclave (Microreactor, Yanzheng Instrument Ltd. Shanghai) and heated in the oven at 200 °C for 7 h. After cooled down in air, the solid product www.nature.com/scientificreports/ was separated by centrifugation, washed with deionized water and absolute alcohol several times, and dried in a vacuum at 60 °C for 6 h.
Reduction and nitridation of Ag-TiN/SMS 39 . The precursor of Ag-TiO 2 /SMS was kept in a horizontal quartz furnace. A flow of N 2 (99.999%) with a rate of 100 mL min −1 was introduced to remove air and moisture for 30 min. Then the furnace was heated from room temperature to 450 °C at a rate of 20 °C ⋅min −1 and the Ag-TiO 2 /SMS was annealed for 1 h. After the furnace temperature was sequentially heated from 450 °C to 850 °C, the flowing gas was switched to NH 3 (160 mL min −1 ) and the nitriding reaction was carried out for 2 h. Finally, the Ag-TiN/SMS were cooled via purging nitrogen gas.
Preparation and characterization of Ag-TiN/SMS electrode. GC (φ = 3.0 mm, S = 0.0707 cm 2 ) was polished by aluminum oxide powders(300 nm and 50 nm respectively), and subsequently washed with acetone, ethanol and deionized water successively for several times. 2.0 mg Ag-TiN/SMS mixed with 100 μl deionized water, 100 μl absolute ethanol and 10 μl 5%Nafion as mixture, and the mixture was sonicated for 30 min. The Ag-TiN/SMS electrode for H 2 O 2 detection were prepared as follows: 3.5 μl the mixture was dropped on the surface of GCE and waited to dry in ambient air. The morphology of TiN/SMS and Ag-TiN/SMS were displayed by scanning electron microscope (SEM), high resolution transmission electron microscopy (HR-TEM) and high angle annular dark field scanning transmission electron microscopy (HAADF-STEM). In addition, analysis of chemical elements in materials was demonstrated by energy dispersive X-ray spectrometer (EDX), using Cu-Kα radiation and spherical-aberration corrected field-emission TEM (Philips-FEI, Tecnai G2 F30 S-Twin). The crystalline structure of TiN/SMS and Ag-TiN/ SMS were analysed by X-ray diffractometer (XRD, PNAlytical),using Cu-Kα as X-ray source and scaning in the range of 20°-80°. The oxidation states of Ag-TiN/SMS were detected by X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra DLD) using a focused monochromatized Al-Kα operated at 300 W. The binding energies were referenced to the C1s line at 284.6 eV from adventitious carbon.

Electrochemical property and amperometric response to H 2 O 2 . Electrochemical property meas-
urements were demonstrated on Ivium potentiostat in 0.02 M PBS (pH 7.0) with different concentrations of H 2 O 2 .The detection of H 2 O 2 by cyclic voltammetry using a three-electrode cell such as the Ag-TiN/SMS as working electrodes, Pt foil as counter electrode and Ag/AgCl as reference electrode. The electrochemical impedance spectroscopy (EIS) was detected by applying 5.0 mV amplitude at a frequency of 100 kHz to 10 MHz. In order to research the selectivity, long-term stability, reproducibility and repeatability of Ag-TiN/SMS. This paper adopted chronoamperometry to compare the response current. The chronoamperometry were also performed in 0.02 M PBS (3.0 mL, pH 7.0) at − 0.3 V. Real sample detection was attested by adding different concentrations of H 2 O 2 solutions to the pre-treated milk sample (3.0 mL, pH 7.0).

Results and discussion
Characterization of Ag-TiN/SMS. Figure (Fig. 3c,d). Silver remains highly dispersed over the surface of the submicrospheres.    Fig. 6c, the reduction peak current increased with the increment of the scan rates in the range of 20-100 mV s −1 . Besides that, there is a linear relation between the square root of scan rates and the reduction peak currents shown in Fig. 6d, indicating that the process is also probably diffusion controlled, which is perfect for quantitative determination. According to the formula, the diffusion coefficient (D 0 ) and reaction rate constant (k 0 ) are calculated by Eqs. (1), (2) 40 .  Figure 6e is the relation of lnI p and (E p − E 1/2 ) at Ag-TiN/SMS, which shows linear section with the linear relationship of lnI p = -11.608 (E p − E 1/2 ) − 11.945 (R 2 = 0.993). The calculated k 0 value on Ag-TiN/SMS electrode is 2.10 × 10 -6 cm s −1 .
To better illustrate the relative enhancement of the catalytic activity on Ag-TiN/SMS, electrochemical impedance spectroscopy (EIS) is carried out under the same experimental conditions to investigate the interfacial properties of TiN/SMS and Ag-TiN/SMS electrodes. The obtained Nyquist plots are shown in Fig. 7. The parameters (1) I p = 2.99 × 10 5 n 3/2 α 1/2 AC 0 D 1/2

Detection performance of Ag-TiN/SMS towards H 2 O 2 . Amperometric I-t curves were performed
with the successive addition H 2 O 2 into a stirring electrochemical cell containing 3 mL PBS (0.02 M, pH 7.0) at an optimized potential of − 0.3 V (Fig. 8a). The inner diagram in the figure is an enlarged version of the 0-400 s. For Ag-TiN/SMS electrode, each response current step showed a smooth trend between 0.5 and 2100 μM. It means that Ag-TiN/SMS electrode can quickly reach a stable response current over a wide range of concentrations. This may be the stability of the electrode material was improved after nitriding. And the electron transfer rate of the Ag-TiN/SMS is increased during the nitriding process. Figure 8b shows the linear fitting relationships between the current responses with different concentrations of H 2 O 2 . To regress from the I-t tests results for Ag-TiN/SMS electrodes. Their current responses as functions of H 2 O 2 concentration can be represented by three different linear equations, which are valid at different There results demonstrate that Ag-TiN/ SMS provides a facile but effective method to fabricate high-performance electrode in sensing applications. Compare the reports of various hydrogen peroxide sensors, as shown in Table 2, the Ag-TiN/SMS exhibited the lowest detection limit with good linear range and the fast-current response towards H 2 O 2 . Perhaps in the composite, titanium nitride may play an important role as a substrate led to the response time of the Ag-TiN/ SMS about hydrogen peroxide was significantly shortened and the electron transfer rate goes up.  www.nature.com/scientificreports/ Selectivity, long-term stability, reproducibility and repeatability of Ag-TiN/SMS. In the process of electrochemical detection, the interference study is a very important aspect in the determination of any species. To detect selectivity of Ag-TiN/SMS, the modified electrode was evaluated in the presence of common interfering electroactive substances such as vitamin C (VC), uricacid (UA), L-glucose (L-Glu), glycine (Gly) and lactosum (Lac) in PBS. As shown in Fig. 9a, the current responses about 0.5 mM vitamin C, uricacid, L-glucose, glycine and lactosum were negligible when compared with 0.1 mM H 2 O 2 . Thus, Ag-TiN/SMS electrode exhibited highly selectivity for H 2 O 2 detection. The long-term stability of sensor was investigated over a 30-day period (Fig. 9b). The current response to 0.5 mM of H 2 O 2 maintained about 91.67% of the original value after the storage period of 30 days. It follows that TiN/SMS substrate material can help silver nanoparticles grow uniformly and contribute to the good stability.
To study the sensor reproducibility, eight Ag-TiN/SMS sensors were prepared by the same method and tested for the H 2 O 2 (0.5 mM) under the same condition (Fig. 9c). The relative standard deviation (RSD) of the response on these eight electrodes is 1.9% by calculation, showing an acceptable reproducibility. Moreover, the RSD of the response repeated for five succeeding measurements is 1.5%. Obviously, the proposed Ag-TiN/SMS electrode demonstrated outstanding repeatability (Fig. 9d).

Real sample analysis.
To investigate the potentials of the sensor to real samples, the Ag-TiN/SMS was evaluated. Different concentrations of H 2 O 2 solutions were prepared using the diluted milk sample. According to FDA, the concentration of H 2 O 2 in milk samples should be less than 14.6 μM 40 . Hence, in order to further explore the possible effectivity of the developed sensor to real sample analysis, various concentrations of these solutions were added to the electrochemical cell containing 3 mL PBS and the amperometry responses were recorded. As is listed in Table 3, the recovery was in the range of 97.00-102.01%, suggesting that the proposed sensor can be applied to detection of H 2 O 2 in practical. The milk sample without H 2 O 2 did not show any detectable signal.

Conclusions
In brief, the Ag-TiN/SMS was successfully prepared by one-pot solvothermal reaction and subsequent nitridation, and then directly applied in a non-enzymatic electrochemical determination of H 2 O 2 . The Ag-TiN/SMS exhibited the excellent catalytic activity towards H 2 O 2 . Electrochemical experiment results show that the presence www.nature.com/scientificreports/ of Ag 0 and TiN/SMS were both responsible for the greatly enhanced performance of sensor. The fabricated Ag-TiN/SMS electrode shows high reproducibility, great analytical selectivity, sensitivity, and stability, making it one of the promising candidates for efficient and sensitive determination of H 2 O 2 . Furthermore, amperometric characterization revealed that the developed non-enzymatic electrochemical sensor for detection of H 2 O 2 from 0.05 to 2100 μM was effective, and the detection limit can reach as low as 7.7 nM (S/N = 3). For real samples, the fabricated sensor also reliably applied in detection of H 2 O 2 at milk. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.