The structural shape of graphene based nanocomposites incorporated with polymers as well as metallic nanoparticles, and other species to harvest synergetic effects and integrated properties of the individuals, has claimed great interest due to their wide range of scientific as well as industrial implications (Badhulika et al., 2015; Sun et al., 2016; Liao et al., 2018; White et al., 2019; Gao et al., 2021). Graphene has, for a long time, been an attractive choice as the building block for nanocomposites owing to its extraordinary mechanical, electronic, and thermal characteristics, as well as the large surface area (Sun et al., 2015; Zeng et al., 2019; Zhao et al., 2020). Graphene nanocomposites with well-designed nanostructures have been extensively fabricated in order to produce diverse materials with desired performance (Aziz et al., 2019a; Asif et al., 2019; Ibrahim et al., 2021). In particular, graphene and metal based nanoparticle composites have been an area of immense significance from the present day research perspective taking advantage of their unmatched implications in the area of sensors, fuel cells, and energy conversions (Nan et al., 2018; Darabdhara et al., 2019). So far, huge progress is reported to have been undertaken for the improved synthesis as well as manufacturing of graphene and metal based nanoparticle composites (Huang et al., 2011; Mangadlao et al., 2017; Ashraf et al., 2020). Among them, graphene-gold nanoparticles (Au NPs) are the ones being investigated the most, and many systematic approaches are reported to prepare Au NPs with various sizes (Asif et al., 2018a; Li et al., 2021). However, some challenges still remain in those methods, such as how to prevent the aggregation and restack of graphene sheet to get higher specific surface areas; how to ensure improved biocompatibility; and how to keep in check the spatial distribution as well as size for the nanoparticles on the surface of graphene. To address those problems, a surface modification strategy of graphene is proposed to fabricate an efficient substrate for the development and growth of gold NPs.

Covalent binding of polymers with graphene through interfacial interactions represents an effective approach for the development of special functional graphene composite (Sun et al., 2020; Perumal et al., 2021). When incorporated with polymer, the aggregation of graphene nanosheets will be effectively inhibited, thus providing the large surface area (Sun et al., 2017). In this paper, covalently functionalized graphene sheets are prepared through grafting poly acrylic acid (PAA) chains on its surface. PAA is a water soluble polymer with many advantages, such as good biocompatibility, non-toxicity, and easy chemical modification (Kausar, 2020). Both hydrophilicity and biocompatibility of graphene could be realized by functionalization of graphene with PAA. Because of plethora carbonyl groups on PAA, dopamine (DA) can be covalently bonded to PAA chain to form PDA-PAA-graphene. Dopamine, a naturally occurring neurotransmitter, found in a variety of animals, is generally regarded as a unique component that can mimick the proteins which are adhesive in nature (Li et al., 2015). Moreover, DA is also used as a reducing agent and stabilizer which is commonly used for the synthesis of metal-nanoparticles (Au, Ag, Pt et al.) by directly reducing the salt of metal precursor (Zhang et al., 2013; Ren et al., 2016; Wen et al., 2018). By using the in situ reducing method, Au NPs could easily be distributed upon the surface of the graphene nanosheets in a homogeneous way. The resulting Au NPs-PDA-PAA-graphene nanocomposites work an ideal platform for the modification of biomolecules. Horseradish peroxidase (HRP), with this unique ability of catalyzing the oxidation for an ample amount of substrates via hydrogen peroxide or any other closely related components, is hailed as a mainstream tool metalloenzyme for establishing the electrochemical biosensors (Sellami et al., 2022). As a potential application, by further linking peroxidase, an amplified biosensing toward H₂O₂ at such Au NPs-PDA-PAA-graphene nanocomposites is constructed.

H₂O₂ is reckoned as an extremely stable reactive oxygen species, which serves as a transporter in a variety of ongoing cellular activities, which holds a pivotal contribution in the case of dealing with unwanted oxidative stress, slowing down the aging process, as well as combating the pathological processes, like cancer, neurological disarray, and cardiovascular issues (Xiao et al., 2012; Asif et al., 2017; Zhang et al., 2017; Asif et al., 2018b; Aziz et al., 2019b; Zhao et al., 2021). Therefore, the quantitative determination of intracellular and extracellular H₂O₂ is known to offer more comprehensive illustration of the clinical aspects of the rise in H₂O₂ concentrations, alongside its assistance in studies put forward to elaborate the biological implications of H₂O₂ in cells (Xi et al., 2018; Ma et al., 2020; Yuan et al., 2020; Zhang et al., 2020; Wang et al., 2021; Zong et al., 2021).

In this paper, a novel Au NPs-PDA-PAA-graphene composite film is designed, which offers superior results both in loading and in the activity of immobilized enzymes. Apart from this, the mentioned film also presents improved electrocatalysis/nano-enhancement activity for H₂O₂ biosensing. PAA as a “friendly” and “soft” linker, DA as a coupling and reducing agent for HAuCl₄, Au NPs as a catalyst for electrode reaction, and HRP as an enzyme, are mixed to yield graphene-based bionanocomposites. The proposed material showcases impressive biocompatibility, superbly uniform distribution of the nanoparticles, and a much higher loading/activity of the immobilized enzymes. This nanohybrid system shows immensely bettered amperometric biosensing activity in contrast to those based on traditional methods and techniques, e.g., a higher detection sensitivity or a sub-micromolar level detection limit, which makes H₂O₂ extremely useful for both in vivo and in vitro detections. This graphene-based bionanocomposites enzyme electrode shows great potential application in the second-generation style of biosensing.

The detailed process employed for carrying out the preparation of the Au NPs-PDA-PAA-graphene composite is illustrated in Figure 1. The synthetic procedure of PAA-graphene was reported previously (Sun et al., 2018), then DA is covalent linked to PAA via the condensation reaction between the NH₂ group in DA and the carboxyl group in PAA, meanwhile, DA will polymerize to form polydopamine (PDA) in alkaline conditions. Next, Au NPs are immobilized onto PDA-PAA-graphene by in situ reducing of HAuCl₄ by dopamine. Au NPs-PDA-PAA-graphene was coated on the glassy carbon electrode to build a platform for enzyme loading. Finally, HRP was absorbed on the electrode to construct an enzyme electrode.

X-ray photoelectron spectroscopy (XPS) is first applied to trace the synthesis process. The condensation reaction between dopamine and PAA-graphene can be verified by noticing a new absorbance peak at 400.8 eV linked to the binding energy of N1s in an XPS spectrum of PDA-PAA-graphene (Figure 2A). After introducing Au NPs to PDA-PAA-graphene, the representing peaks of Au 4f_7/2 and Au 4f_5/2 show up at the binding energies of 83.9 and 87.7 eV respectively (Figure 2B), suggesting the successful immobilization of Au NPs upon the PDA-PAA-graphene surface. FT-IR measurement provides further evidences of the above chemical changes as the carboxylate peak in PAA is no longer present, and amine and aromatic rings peaks appear at 1,670, 1,620, and 1,550 cm⁻¹, individually (Figure 2C) (Wu et al., 2011).

The condensation reaction can also be identified by 1H NMR spectrum (Figure 2D). Comparing with that of PAA-graphene, new peaks appear at d 6.83–6.40 and 2.5–3.0 at the 1H NMR spectrum of PDA-PAA-graphene, which directly corresponds to the chemical shift of protons in aromatic rings and alkyl of DA. f = A/A₀ is used to determine the dopamine content (mol%) in PDA-PAA-graphene. Where, A stands for the integral part of the peaks at d 6.83–6.40 representing the quantity of H in the aromatic rings of the given catechol moieties, while A₀ corresponds to the integral part of the peaks at d 2.2–1.2 showcasing the quantity of H in the PAA backbone. The dopamine ratio found in the resultant polymer is nearly 25% as per the given method. This result is a little lower than that calculated by thermogravimetric analysis (31%) (Supplementary Figure S1).

The as-prepared Au NPs-PDA-PAA-graphene composite is also confirmed by UV-vis absorption spectrum as displayed in Figure 2E. The spectrum of PAA-graphene presents a maximum absorption at 263 nm, which signifies π-π* transitions between the aromatic C=C bonds (He et al., 2011). After reacting with DA, a shoulder peak at 275 nm, a characteristic absorption for catechol, is detected (Bui et al., 2021). After reduction of HAuCl₄, a relatively new peak at 556 nm appears and the solution becomes violet black in color. This newly appearing peak can well be stemming from the surface plasmon resonance (SPR) absorptive band for Au NPs, highlighting the reducibility of AuCl₄ ⁻¹ to Au NPs in a successful way (Mawlud et al., 2017). In contrast to the as-prepared Au colloids present in aqueous solution (Song et al., 2011), the obtained absorption maxima is found to be red-shifted, which in turn can consequently be attributed to the altering refractive index and to some extent partial aggregation of the seed particles inside the growth solution. These results further go on to prove that as far as in situ methods are concerned, Au nanoparticles are closely linked with each other for the sake of the reduction process and also to small quantities of agglomerations or even bigger particles are expected to have deposited on the graphene surface. The crystalline XRD pattern of Au NPs-PDA-PAA-graphene nanohybrid (Figure 2F) show a peak at 24.3 indexed to the (002) crystal face of graphene, and the peaks at 38.1, 44.3, 64.5, and 77.6 correspond to the (111) (200), (220), and 311) crystal faces of Au nanoparticles (JCPDS 4–0,783) (Li et al., 2012). Quantitative measurements of Au NPs on Au NPs-PDA-PAA-graphene surface are materialized by EDX, pointing out the presence of 58 wt% of Au NPs (Supplementary Figure S2).

SEM and TEM are used to undertake the characterization of the morphology of the Au NPs-PDA-PAA-graphene composites. Figures 3A,B, represent the random distribution of Au NPs PDA-PAA-graphene surface. These also show the fact that the nanoparticles are well embedded on the interior of the surface, highlighting a close interaction among the NPs and PDA-PAA modified graphene sheets. The distribution of Au NPs is believed to be relatively uniform with no severe aggregations. TEM images signify samples to be composed of nanoparticles having diameters averaging nearly 18 nm and the size distribution ranging between 5 and 30 nm. The diameter of most of the found particles is between 12 and 20 nm (Figures 3C,D).

The novel nanostructure of the Au NPs-PDA-PAA-graphene inspires us to investigate it as enzyme biosensor. Figure 4 is the CV representation of differently modified electrodes in 50 mm PBS (pH = 7.4), with 50 mV s⁻¹ scan rate. In the CVs of Au NPs-PDA-PAA-graphene and PDA-PAA-graphene electrodes, no observable redox peak appeared which is indicative of their electrochemical inactivity at the specific potential range (−0.8 + 0.1 V). A clearly distinguishable couple of redox peaks in the CVs of HRP/PDA-PAA-graphene are attributed to the Fe^III/Fe^II redox coupling related to the heme protein for HRP (Liu et al., 2012). While in the CV curve of HRP/Au NPs-PDA-PAA-graphene, the Fe^III/Fe^II redox couple is more distinct with the cathodic peak potential (−0.320 V) and anodic peak potentials (−0.267 V). Considering the conventional potential [E_o’ = (Epa + Epc)/2] of −0.294 V, and peak point separation (ΔEp) ∼53 mV at the scan rate of (50 mV/s), indicating a reversible electrochemical behavior of HRP, which is much better than other HRP-based biosensors known so far (Zong et al., 2006; Tang et al., 2008). This phenomenon is the proof of uninterrupted transfer of electrons between HRP and GCE, initiated by Au NPs as a promoter. The chemistry behind the direct electron transportation between the HRP and GCE is based on the Au NPs, which not only reduce the space for the center of activation for the immobilized HRP and the GCE, but also effectively boost up the electrochemical conductance of the fabricated electrode, in turn, encourages direct electron transportation between the HRP and glassy carbon electrode. As the active sites of HRP mostly buried deep inside the center and make it harder to exchange electrons with the electrode surface (Li et al., 2011), so Au NPs act as a mediator to carry out the fast electron transfer rate which in turn greatly enhanced the electrochemical performance of the synthesized electrode.

To further clarify the catalytic activity between the HRP and the electrode we used the Au NPs-PDA-PAA-graphene modified electrode immobilized with HRP to study the thoroughgoing current response by applying a different scan rate. As represented in Figure 5A, although the redox current gradually increased with an increase in the scan rate, the peak potential did not change obviously. Figure 5B shows the excellent linear relation between the redox peak current values and increased scanning rate, ranged from 0.1—1 V/s. Here the regression equation; i p a = 1.00 × 10 - 4 + 0.122 v ( R = 0.999 ) i p c = - 9.33 × 10 - 5 - 0.103 v ( R = 0.999 )

Here, v represents the scan rate while i _pa, i _pc are the peak current density of anode and cathode. This suggests that the redox reaction of HRP is a typical facet constrained reversible phenomenon. With the assimilation of current (reduction peak) and Faraday’s law, the electrochemically active HRP can be calculated from HRP/Au NPs-PDA-PAA-graphene surface from the equation. (Zhang et al., 2010), i p a = n 2 F 2 A Γ ν 4 R T where ipa signifies the reduction peak current, n denotes the total amount of electrons, A stands for the electrode surface area, v is the scan rate, and Γ is the value which needs to be calculated. The estimated Γ value was 9.57 × 10⁻¹¹ mol cm⁻². The Au NPs-PDA-PAA-graphene composite matrix gives remarkable higher surface concentration value for the immobilization of HRP as compared to the previously published literature using different types of matrix, such as colloidal Au (7.5 × 10^–11 mol cm⁻²) (Liu and Ju, 2002)^], 3-mercaptopropionic acid monolayer-modified gold surface (5 × 10⁻¹¹ mol cm⁻²) (Li and Dong, 1997).

H₂O₂ has been used as an analyte to study the catalytic applications of the fabricated HRP/Au NPs-PDA-PAA-graphene films. Figure 6 explained the catalytic behavior of the fabricated material in the presence and absence of H₂O₂. When the different concentrations of H₂O₂ were injected into PBS, there was a clear rise in the reduction peak current with a gradual decrease in the oxidation peak current. This phenomenon well explains the remarkable electrocatalytic process for the HRP oxidation via H₂O₂ which in turn was reduced. H₂O₂ oxidized the HRP_re to HRP_ox which was further reduced through electron transfer upon the electrode surface. As H₂O₂ concentration increased it encouraged a large cathodic peak current value, in turn there was a fast rate of electron transfer between the quantities of electrode surface and immobilized HRP.

The amperometric response of the HRP/Au NPs-PDA-PAA-graphene film electrode with sequential injection of H₂O₂ in PBS at −0.32 V is shown in Figure 7. The fabricated electrode gives a quick response towards any change in the H₂O₂ concentration and attains a steady state within 5 s, which is credited to the rise in electron transfer from the embedded Au NPs. The lowest limit of detection (0.02 μM S/N = 3) and an excellent linearity range from 0.1 μm to 20 mm (R = 0.999) is better than that in similar systems previously reported (Lin et al., 2007; Lu et al., 2011; Liu et al., 2012). These results suggest that HRP/Au NPs-PDA-PAA-graphene nanoarchitectures pave the way for the synthesis of enzymatic biosensors for the sensitive detection of H₂O_2.

The repeatability parameter was evaluated by taking 10 repeated measurements with the consecutive amount of 1.0 mm H₂O₂ with a same electrode which produces relative standard deviation (RSD) of ∼3%. Furthermore, the RSD for the current responses for more than 10 sensors obtained by following the similar protocol is ∼5%. For the stability evaluation of the HRP/Au NPs-DA-PAA-graphene film electrode preserved at 4°C, it gives 90% response even after 2 months of refrigeration (Supplementary Figure S3). The application of Au NPs-PDA-PAA-graphene composite enhances the biocompatibility and provides a favorable microenvironment for HRP, which aids the retention of the biological activity of HRP. Meanwhile, we speculate that the hydrophilic character of Au NPs-PDA-PAA-graphene would also improve the HRP catalytic performance it does not gets denaturation as well as no reduction in catalytic functioning has been observed at low temperature. Conversely, the sensor sensitivity get reduced quickly at room temperature which may be the reason for the swift deactivation of HRP.

By keeping in mind all the advantages of the excellent catalytic performances of the fabricated electrode such as lowest limit of detection, enhanced sensitivity, and quick response, it is noteworthy to say that the as-fabricated biosensor can be used for the determination of the extracellular H₂O₂ released by macrophage cells. The used macrophages offer a pivotal role against the different kind of microorganisms and tumors in there host cells (Niedel et al., 1979). However, these two special properties (tumoricidal and microbicidal) of the chemotactically responsive macrophages required some types of agents like bacterial products, cytokines, synthetic peptide, and some chemotherapeutic drugs for their activation. The stimulator N-formyl-methionyl-leucyl-phenylalanine (fMLP) used in this work is a synthetic peptide which is structurally identical to oligopeptide products of bacterial metabolism (Holian and Daniele, 1981). The chemistry behind the use of fMLP as a stimulator is not only its chemotactic properties but it also facilitates the adhering of PMN to endothelium cells. Moreover it also stimulates the release of many kind of molecules such as reactive oxygen intermediates (ROI), lysosomal enzymes, nitric oxide, interleukin 1 (IL-1), and tumor necrosis factor. In this work, the cell macrophages regarding 80% confluency are subjected to release H₂O₂ after injecting fMLP. In the experiment of real-time sensing of H₂O₂ secretion by macrophages, calcein-AM is used to stain the live cells. The dark field microscopic images refer to the fact that the stained macrophages cells are well-spread and healthy (Figure 8A). According to the experimental results, after the addition of each 10 μm concentration of fMLP a clear current increase (2.8 μA cm⁻²) has been observed on the HRP/Au NPs-PDA-PAA-graphene film electrode at the applied potential (−0.32 V), which then further decreases as the H₂O₂ scavenger catalase was spiked. The same procedure was carried out with control well plates (without macrophages) which showed no response as depicted by Figure 8B). Therefore, we can conclude that the reported rise in cathode current at HRP/Au NPs-PDA-PAA-graphene film electrode placed near the cells is endorsed to H₂O₂oxidation. All the above discussed results markedly validate that the as-fabricated biosensor can initiate a new opening in biosensing platforms for the unswerving determination of extracellular as well as intracellular H₂O₂ and could well be effectively helpful for advanced physiological and pathological related studies.

In summary, new Au NPs-PDA-PAA-graphene composite films have been synthesized by loading highly active enzyme immobilization. The material is introduced onto the electrode surface, and serves as biosensor for extracellular and intracellular H₂O₂ detection. The biosensor exhibits remarkable results in terms of extraordinary sensitivity, wider linear range (0.1 μm–20 mm), noble stability, lowest limit of detection (0.02 mm), and reproducibility for H₂O₂ detection, which is superior to previously constructed biosensing podiums. Hence, this work brings up an advanced biosensing platform for the determination of H₂O₂ in vivo and in vitro, which can be of utmost advantage for bioelectroanalytical chemistry, cellular biology, and pathophysiological studies.