Preparation of graphene nanocomposites from aqueous silver nitrate using graphene oxide’s peroxidase-like and carbocatalytic properties

The present study evaluates the role of graphene oxide’s (GO’s) peroxidase-like and inherent/carbocatalytic properties in oxidising silver nitrate (AgNO3) to create graphene nanocomposites with silver nanoparticles (GO/Ag nanocomposite). Activation of peroxidase-like catalytic function of GO required hydrogen peroxide (H2O2) and ammonia (NH3) in pH 4.0 disodium hydrogen phosphate (Na2HPO4). Carbocatalytic abilities of GO were triggered in pH 4.0 deionised distilled water (ddH2O). Transmission electron microscope (TEM), scanning electron microscope (SEM), cyclic voltammetry (CV) and UV-Vis spectroscopy aided in qualitatively and quantitatively assessing GO/Ag nanocomposites. TEM and SEM analysis demonstrated the successful use of GO’s peroxidase-like and carbocatalytic properties to produce GO/Ag nanocomposite. UV-Vis analysis indicated a higher yield in optical density values for GO/Ag nanocomposites created using GO’s carbocatalytic ability rather than its peroxidase-like counterpart. Additionally, CV demonstrated that GO/Ag nanocomposite fabricated here is a product of an irreversible electrochemical reaction. Our study outcomes show new opportunities for GO as a standalone catalyst in biosensing. We demonstrate a sustainable approach to obtain graphene nanocomposites exclusive of harmful chemicals or physical methods.


Synthesis of GO/Ag nanocomposite using GO's peroxidase-like catalytic properties. GO
surroundings were tailored to stimulate its peroxidase-like catalytic properties 29 and facilitate the oxidation of aqueous AgNO 3 to Ag nanoparticles. Therefore, GO/Ag nanocomposite synthesis was achieved by changing the concentration of GO in the presence of AgNO 3

Synthesis of GO/Ag nanocomposite using GO's intrinsic or carbocatalytic abilities. The need
for Na 2 HPO 4 , H 2 O 2 , and NH 3 to create GO/Ag nanocomposite was assessed by lowering Na 2 HPO 4 concentration (25 mM, 10 mM, 5 mM, 2.5 mM, 0.5 mM, and 0 mM) in the presence of 40 µg GO, 3.2 mM AgNO 3 , 100 mM H 2 O 2 , and 40 mM NH 3 in total 1 ml solution. Particularly, the 0 mM Na 2 HPO 4 (i.e., ddH 2 O) experiment was performed with 40 µg GO and 3.2 mM AgNO 3 in the presence and absence of 100 mM H 2 O 2 and 40 mM NH 3 at pH 4.0 and 37 °C. Without Na 2 HPO 4 , H 2 O 2 , and NH 3 , GO will depend on its natural abilities 25 to convert aqueous AgNO 3 to Ag nanoparticles.
Experimental setup, characterization, and data analysis. In the case of experimental controls, all reaction components were included except AgNO 3 to control for GO and vice versa. Thus, experimental controls have been referred to as GO alone or AgNO 3 alone hereon. The reaction temperature for all experiments was maintained at 37 °C using a heat block, and each component was added at 15 min intervals 29 . The order in which each chemical was added to Na 2 HPO 4 buffer was GO, H 2 O 2 , AgNO 3 , and NH 3 . Further, UV-VIS spectral analysis (Perkin Elmer Lambda 650) was performed between 300 nm to 700 nm 15 min after adding the last element. Specimens for changing GO and AgNO 3 were performed in triplicates to measure average standard deviation across all wavelengths from 300 nm to 700 nm.
Following equation was applied to measure the difference in absorbance between GO/Ag nanocomposite to GO or AgNO 3 alone, wherein "Nanocomposite" refers to the GO/Ag nanocomposite absorbance at 450 nm and "Control" denotes matching GO or AgNO 3 alone absorbance at 450 nm. The horseradish peroxidase (HRP) enzyme reacts with 3,3' ,5,5'-tetramethylbenzidine (TMB) and is detectable at 450 nm that is extensively used for various biochemical applications 31,32 . As GO can mimic peroxidase-like activity 29 , the nanocomposite-to-control percentage measurements were performed at 450 nm. Equation 1 was adapted from Microsoft Office instructions for calculating percentages using the Microsoft Excel. Further, cyclic voltammetry (CV) was performed to study the molecular electrochemistry between GO and AgNO 3 . Following previously described set-up for CV 33 , an in-house CV was www.nature.com/scientificreports www.nature.com/scientificreports/ created using glassy carbon electrodes as the working and counter electrodes with an Ag/AgCl reference electrode (66-EE009) from Cypress systems. A Keithley electrometer series 2400 was utilized together with a homebuilt LabView measurement program to measure current (mA) between the working and counter electrodes, as well as difference in potential (V) between the working and reference electrodes. To avoid interference from dissolved O 2 , the electrolyte solution was purged with N 2 gas. In all CV measurements, ±3 V was applied with a step size of 10 mV and scan rate of 38 mVs −1 .
Visualization studies were conducted to view the nanocomposite structures and aid in elemental analysis. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) required 5 μl of sample on Formvar/carbon coated 200 mesh copper grids. In the case of TEM, digital micrographs were acquired with Jeol JEM-1400HC (Jeol, Tokyo, Japan) that is equipped with Quemesa (Olympus Soft Imaging Systems, Münster, Germany) bottom mounted CCD-camera. Chemical characterization for GO/Ag nanocomposite was achieved with Zeiss EVO-50XVP SEM that is integrated with Bruker Quantax 400 energy dispersive spectrometer (EDS). Elemental maps from EDS were obtained using Bruker AXS detector 3001 with energy resolution <133 eV (MnKa, 1000 cps). The counts per second values in elemental maps were normalized. Image J (https://imagej.nih. gov/ij/ version 1.51) was utilized for measuring the average size of Ag nanoparticles on GO and adding a scale bar.
The nanocomposite-to-control percentage (Fig. 1C) at 450 nm between GO/Ag nanocomposite to matching AgNO 3 alone rises from 47% (40 µg GO/0.4 mM AgNO 3 ) to 77% (40 µg GO/12.8 mM AgNO 3 ). Therefore, varying AgNO 3 amounts in the presence of 40 µg/ml GO yields spectra that are GO/Ag nanocomposite dominant (Fig. 1B). Additionally, Fig. 1B reveals that 40 µg/ml GO/12.8 mM AgNO 3 nanocomposite spectra is the upper limit for detection as the said GO/Ag combination saturates UV-VIS spectroscopy machine's capability to absorb. Hence, the maximum nanocomposite-to-control percentage between GO/Ag nanocomposite to GO or AgNO 3 alone can be either observed at low GO quantities (5 µg GO/0.1 mM AgNO 3 ) or at high AgNO 3 concentration (40 µg GO/12.8 mM AgNO 3 ) as depicted in Fig. 1C. Overall, the absorbance values between 300 nm to 700 nm for all GO/Ag nanocomposites created by varying GO and AgNO 3 concentrations (Fig. 1A,B) were replicable when performed in triplicates with an average standard deviation of 0.008 a.u. and 0.039 a.u., respectively (Fig. S1).
Electron micrograph images of GO (160 µg/ml) or AgNO 3 (12.8 mM) alone ( Fig. S2) were visually compared with images of GO/Ag nanocomposites (Fig. 2). The black dots or cloud-like deposits on GO sheets in Fig. 2 are Ag nanoparticles in comparison to the clean GO sheets in Fig. S2A. Silver nanoparticles were observed exclusively on GO sheets indicating that GO plays an imperative role in oxidizing aqueous AgNO 3 and act as a substrate on which the Ag nanoparticles can nucleate (Fig. 2). Although changing GO concentrations in the presence of 0.1 mM AgNO 3 produce spectra that are GO dominant (Fig. 1A), silver nanoparticles can still get deposited on GO sheets ( Fig. 2A-F). Nucleation of Ag nanoparticles on GO sheet was not restricted and the resulting anisotropic nanoparticles measured larger than 500 nm ( Fig. 2A-F). On increasing the amounts of AgNO 3 , the continuous growth of Ag nanoparticles into colloidal cloud-like particles on GO sheets was noted ( Fig. 2G-L).
Characterization of GO/Ag nanocomposite produced using GO's intrinsic or carbocatalytic abilities. GO can utilize its peroxidase-like catalytic property to oxidize aqueous AgNO 3 to Ag nanoparticles (Figs. 1A,B, and 2). Nonetheless, the nanocomposite-to-control percentage at 450 nm between GO/Ag nanocomposite to GO or AgNO 3 alone is limited to 77% (Fig. 1C). Growing the difference in absorbance between GO/ Ag nanocomposite and GO or AgNO 3 alone at 450 nm should elevate the nanocomposite-to-control percentage (Eq. 1). To assess the reagents that produce high noise, absorbance values of 25 mM Na 2 HPO 4 , 100 mM H 2 O 2 , and 3.2 mM AgNO 3 were collected at 450 nm in several combinations (Fig. S3). Individually, 25 mM Na 2 HPO 4 , 100 mM H 2 O 2 , and 3.2 mM AgNO 3 did not exhibit noteworthy absorbance. In contrast, a twelve-fold increase in absorbance was observed when 3.2 mM AgNO 3 was introduced to 25 mM Na 2 HPO 4 (Fig. S3). An additional three-fold rise in absorbance was noted when 100 mM H 2 O 2 was subjected to 3.2 mM AgNO 3 in 25 mM Na 2 HPO 4 .
Electrochemical behaviour and elemental analysis of GO/Ag nanocomposite. The electrochemical measurements between GO and AgNO 3 in Na 2 HPO 4 or H 2 O yielded cyclic voltammograms with a large capacitive current contribution due to the working electrode having a large surface area. A redox response is most visible in the presence of Na 2 HPO 4 with an oxidation peak at 0.15 V and a reduction peak at −0.1 V (Fig. 3C). GO and AgNO 3 in H 2 O however displays no visible redox response and GO/Ag nanocomposite seems to be fully stable (Fig. 3D). When H 2 O 2 and NH 3 is removed, the CV response shows a broad oxidation curve that suggests a sluggish electron exchange between GO and AgNO 3 (Fig. 3E). In contrast, no discernible oxidation (anodic) or reduction (cathodic) curves were observed when GO or AgNO 3 alone were dissolved in H 2 O (Fig. S4).  3 , and (C) the resulting nanocomposite-to-control percentage at 450 nanometres (nm) between GO and AgNO 3 together (i.e., experiment) and GO or AgNO 3 alone (i.e., control) from (A,B). All chemical reactions were carried out at 37 °C in pH 4.0 solvents. Absorbance was recorded in arbitrary units (a.u.) from 300 to 700 nm. Solid lines represent absorbance due to interaction between GO and AgNO 3 together (i.e., experiment) versus dashed lines indicates absorbance of either GO or AgNO 3 alone (i.e., control). www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ The result of electrochemical behaviour between GO and AgNO 3 can be seen in the electron micrographs presented in Fig. 4. TEM images visibly indicate the deposition of silver nanoparticles on GO sheets in the absence of Na 2 HPO 4 (Fig. 4B) and H 2 O 2 (Fig. 4C) that is comparable to GO/Ag nanocomposite created in 25 mM Na 2 HPO 4 ( Figs. 2 and 4A). Additionally, the presence of silver nanoparticles on GO sheets was confirmed with SEM and EDS (Fig. 5). Here the GO/Ag nanocomposite appears bright compared to the darker background of formvar. Mapping of elements was performed either on (black star) or around (grey star) the GO/Ag nanocomposite (Fig. 5A-C). Similarly, elemental maps include a black or grey line to correspond with said coloured EDS measurement stars (Fig. 5A.1-C.1). Elemental mapping on GO/Ag nanocomposite (black star and line) consistently demonstrated peaks for Carbon (C at 0.28 keV), Oxygen (O at 0.53 keV), Copper (Cu at 0.93 keV), Aluminium (Al at 1.49 keV), and Silver (Ag at 2.99 keV, 3.17 keV, and 3.33 keV). The EDS measurements around GO/Ag nanocomposite (grey star and line) did not yield any Cu or Ag peaks, while the C and O peaks are consistent with the underlying formvar. Figure 5D,D.1 indicate that GO is the source for Cu, as also seen in GO/Ag nanocomposite elemental maps (Fig. 5A.1-C.1). Further, Al was seen in all EDS measurements due to the sample holder beneath the formvar copper grid. Figure 6 illustrates a comparison between the synergistic and inherent catalytic approaches for creating graphene nanocomposites. Furthermore, Fig. 7 proposes a mechanism to understand the surface chemistry involved in oxidizing AgNO 3 to Ag nanoparticles.

Discussion
We demonstrate the use of GO's peroxidase-like 29 and inherent or carbocatalytic properties 25 to oxidize inorganic compounds such as AgNO 3 . The Na 2 HPO 4 buffer with H 2 O 2 activates the peroxidase-like catalytic property of GO that converts AgNO 3 to Ag nanoparticles ( Figs. 1 and 2). The nanocomposite-to-control percentage for resulting GO/Ag nanocomposite is a maximum of 77% (Fig. 1C) because the interaction between Na 2 HPO 4 and AgNO 3 produces high noise (Fig. S3). In contrast, GO/Ag nanocomposite created in deionized distilled water (pH 4.0) without H 2 O 2 yields 97% in nanocomposite-to-control difference (Fig. 3B). In the absence of Na 2 HPO 4 and H 2 O 2 , GO's intrinsic ability facilitates the transformation of AgNO 3 to Ag nanoparticles (Figs. [3][4][5]. Large aromatic basal planes of GO offer a high surface area containing epoxy, hydroxyl, and carboxyl functional groups that contribute to GO's inbuilt catalytic properties 25 . Our findings indicate that GO alone can oxidize AgNO 3 and also act as a substrate for Ag nanoparticles to generate GO/Ag nanocomposites. The GO/Ag nanocomposite created in this study using only natural catalytic properties of GO is simple, resource-efficient, and swift compared to old techniques (Fig. 6). Traditionally, synthesis of GO nanocomposites with metal nanoparticles as shown in Fig. 6 includes pre-treatment of GO, organic/inorganic solvents to dissolve the metal precursor, and chemical/physical methods to reduce and stabilize the final product [16][17][18][19][20][21][22][23][24] . GO pre-treatment may involve adsorption or electrodeposition of metal ions 16 , conjugation with antibodies 17 , PEG 19 , and more 18,23 . Further, solvents of metal precursors such as AgNO 3 16 , chloroplatinic acid (H 2 PTCL 6 ) 21 , and cobalt (II) acetate (Co(CH3COO) 2 ) 22 include deionized distilled water 19 , ethanol 22 , 1% BSA 17 , citrate buffer 16 , or sulphuric acid 23 . Ultimately, blending of GO with metal nanoparticles demands the presence of chemical agents like hydroquinone 16,17 , hydrazine hydrate 20 , sodium borohydride (NaBH 4 ) 24 , or ammonium hydroxide (NH 4 OH) 22 to reduce and stabilize the metal clusters on GO surface. Alternatively, extreme physical methods such as autoclave 15,22 , annealing or heating at 60 °C to 600 °C 19,20,23 have been reported to create metal nanoparticles over GO.
Catalytic properties of GO are either synergistic or inherent following the literature 15 and our research findings (Figs. 4-6). A synergistic approach advocates the integration of GO with other catalytic components such as silver 16 , palladium 20 , platinum 21 , or cobalt 22 nanoparticles using pre-treated GO [16][17][18][19][20]23 , chemical agents 16,17,[20][21][22]24 , or heating 19,20,22,23 . As a result, GO in synergy with metal nanoparticles participates in photocatalysis 24 , photodegradation 23 , bactericidal activity 16 , Suzuki-Miyaura coupling reaction 20 , oxygen evolution reaction 22 , and methanol 21 or oxygen oxidation reaction 22 . In contrast, the oxidation of AgNO 3 to Ag nanoparticles in this study is influenced by the functional group composition, structure, and morphology of GO (Figs. 3-5) that signifies its inherent catalytic properties 25 . Application of GO's intrinsic catalytic abilities is limited to either hydration or oxidation of various organic compounds, including alcohols, alkynes, and alkenes 34 . Additionally, the use of heteroatom doped GO in oxygen reduction reactions is considered as a natural use of GO's catalytic abilities [34][35][36] . However, attaching heteroatoms like sulphur 36 and iodine 35 to GO also comprises several pre-treatment processes as vigorous ultrasonication, and annealing at 500 °C to 1100 °C in an argon atmosphere 35,36 .
The GO/Ag nanocomposite fabricated here is a product of an electrochemical irreversible reaction (Fig. 3C,D). Kinetics that may govern the irreversible nucleation, growth, and attachment of Ag nanoparticles on GO (Fig. 7) can be described in three steps (1) monomer supplying reaction, (2) growth by diffusion and (3) autocatalytic oxidation 37,38 . Same three steps also explain the development of gold nanoparticles in a classical Turkevich method wherein citrate and squaric or ascorbic acid are used as reducing agents [37][38][39][40][41][42][43] . The monomer supplying reaction (Fig. 7) is imperative for making Ag monomers/ions and clusters 37,38 . Primary contact between AgNO 3 and functional groups of GO may initiate the creation of many Ag monomers over GO. Coalescence follows the creation of Ag monomers due to the low aggregation barrier to form first clusters or Ag seed particles (Fig. 7). Lower or higher aggregation barrier is essentially activation energy that either promotes coalescence among monomers or inhibits aggregation between large nanoparticles, respectively 37,38,40 . In the next step, newly formed Ag monomers may diffuse onto the existing seed particles to facilitate the growth phase of Ag nanoparticles over GO 38,40 . 16.16.3 (181015)), QtiPlot (https://www.qtiplot.com/ version 10.9), Adobe Photoshop 2020 (https://www.adobe. com/in/products/photoshop.html version 21.0.2), and Adobe Illustrator 2020 (https://www.adobe.com/in/ products/illustrator.html version 24.0.1). www.nature.com/scientificreports www.nature.com/scientificreports/ Unlike the Turkevich method 37-42 , the size, shape, density, and distribution of Ag nanoparticles on GO is not homogenous in our study. Nonetheless, heterogenous GO/Ag nanocomposites have produced replicable results when characterized using UV-VIS spectroscopy (Fig. S1). The growth by diffusion step is crucial for narrowing polydispersity among gold nanoparticles that are produced in suspension using the Turkevich method 37-42 . In www.nature.com/scientificreports www.nature.com/scientificreports/ contrast, nucleation and growth of Ag nanoparticles are restricted by GO's finite sheet size (Figs. 2, S2, 5) and island-like arbitrary distribution 44,45 of active sites (i.e., epoxy, hydroxyl, and carboxyl functional groups). Also, the size and density of Ag nanoparticles on GO is determined by the starting quantity of AgNO 3 (Fig. 7). Small amounts of raw AgNO 3 are readily converted to Ag nanoparticles that are distinguishable (Fig. 7A). However, Figure 6. Graphene oxide (GO)/metal nanocomposites can be achieved without the use of harmful chemicals and extreme physical reduction methods. Schematic comparing key requirements to generate GO/metal nanocomposites following reported methodologies (old approach) versus current technique (new approach). www.nature.com/scientificreports www.nature.com/scientificreports/ larger quantities of AgNO 3 are transformed to Ag cloud-like nanoparticles that prominently cover the GO sheets (Fig. 7B). Cloud-like nanoparticles may result due to sizeable AgNO 3 residual that is continuously oxidized to Ag monomers and diffused onto the existing Ag nanoparticles (i.e., autocatalytic oxidation) over a finite size of GO sheet (Fig. 7B).
Among various inorganic compounds, applications of silver nanoparticles in the biomedical field is widespread and ever-increasing due to its low-cost, abundance, and fascinating properties 46,47 . As a consequence, silver nitrate was utilized in this study to embody GO's peroxidase-like and natural or carbocatalytic oxidation capabilities with inorganic compounds. However, as Ag is monovalent, more research is required to understand the limits of GO's peroxidase-like or intrinsic catalytic properties using divalent, trivalent, and tetravalent inorganic elements 48 . The intrinsic and peroxidase-like catalytic properties of graphene shown here may be seen in its allotropes like carbon nanotubes [49][50][51][52] and fullerenes 53 coupled with epoxy, hydroxyl, and carboxyl groups. Also, in future experiments, radical scavenger study and electron spin resonance technique can help understand the influence of free radicals, O 2 , and OH groups (i.e., resulting from decomposed H 2 O 2 ) on the fabrication of carbon nanocomposites.
What plastic was for the 20th century, graphene is to the 21st century. The World Bank and the United Nations have adopted several sustainable development goals to reduce the risk that plastic poses to our environment and public health 54 . Currently, the lack of standardized health and safety guidelines to assess the toxic influence of unique graphene-based technologies on human health and the environment is staggering 14 . In Europe, the registration, evaluation, authorization, and restriction of chemicals (REACH) regulation (EC 1907(EC /2006) by the European Agency for Safety and Health at Work instructs wellbeing at organizations that manufacture or import harmful chemicals 55 . Nonetheless, the REACH regulation only applies to establishments dealing with one tonne or more graphene per year 14 . In 2017, the International Organization for Standards (ISO) published ISO/TS 80004:13:2017 56 that only clarifies technical pre-requisites for a material to be deemed as graphene or otherwise.