Electrochemically reduced graphene oxide modified with electrodeposited thionine and horseradish peroxidase for hydrogen peroxide sensing and inhibitive measurement of chromium

Materials Science for Energy Technologies 2 (2019) 676–686

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Materials Science for Energy Technologies

CHINESE ROOTS GLOBAL IMPACT

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Electrochemically reduced graphene oxide modified with electrodeposited thionine and horseradish peroxidase for hydrogen peroxide sensing and inhibitive measurement of chromium Shailendra Kumar Pandey a, Sadhana Sachan b, Sunil Kumar Singh c a b c

Department of Chemical Engineering, Harcourt Butler Technical University, Kanpur, India Department of Chemical Engineering, Motilal Nehru National Institute of Technology Allahabad, India Department of Animal Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda 151001 Punjab, India

a r t i c l e

i n f o

Article history: Received 7 May 2019 Revised 31 July 2019 Accepted 9 August 2019 Available online 11 September 2019 Keywords: Electrodeposition Electroreduced graphene oxide Peroxide Horseradish peroxidase Chromium Enzyme inhibition

a b s t r a c t Fabrication of hydrogen peroxide (H2O2) biosensor for determination of Cr(VI) based on the inhibition of horseradish peroxidase (HRP) activity is reported herein. The sensor platform was developed by using electrochemically reduced graphene oxide (ERGO) film modified GCE electrode. The thionine layer is deposited over ERGO layer by two step electrodeposition process. Immobilization of HRP over the thionine modified ERGO electrode has been done by mixing with bovine serum albumin (BSA) and using glutaraldehyde as a crosslinker. The cathodic responses of the immobilized HRP for H2O2 detection is monitored using cyclic voltammetry and linear sweep voltammetry method. Cr(VI) as a trace metal ions inhibitor to the enzyme activity was proportional to concentration in the range of 0.039–0.78 mM with the sensitivity of 4.41% inhibition mM
1 and the limit of detection is 0.02 mM (S/N = 3). The inhibition mechanism determined from Dixon and Cornish-Bowden plots was found to be uncompetitive. Good stability and reproducibility of the developed sensor could provide an excellent sensing platform for the determination of peroxide and heavy metals in environmental analyses and clinical testing. Ó 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).

1. Introduction Heavy metals are non-degradable compounds and occur naturally in the ecosystem with large deviations in concentration and contaminate the wastewater through various channels. The extensive released of heavy metal ions from numerous industrial activities leads to serious toxicity for living organisms and economic damage. The runoff of water from different sources from electroplating industries, petroleum refining units, manufacturing process of glass and electronic parts, paints and used batteries are the main cause of heavy metal pollution [1–3]. Heavy metals may cause risk to human health and biologically transfer into our ecological systems & food chains [2,4]. Interaction of these metal ions with enzymes by binding to the sulfhydryl groups, leads to inhibit the

E-mail-address: [email protected], [email protected] (S.K. Singh). Peer review under responsibility of KeAi Communications Co., Ltd.

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function and metabolism of numerous proteins, enzymes and hormones [5]. Chromium is one of the major environmental pollutants released from leather tanning, metallurgic and electroplating industries. Cr(III) and Cr(VI) are the two stable oxidation state of chromium with different toxicity level. Cr(III) is an essential nutrient and comparatively very less toxic and less mobile as compared to Cr(VI) which is highly mutagenic and carcinogenic & having strong oxidizing properties with 500–1000 times more toxic than Cr(III) [6–8]. Hydrogen peroxide (H2O2) is recognized as one of the major factors in various fields including food, textile, pharmaceutical and environmental analyses [9,10]. The sensing of H2O2 is of great importance in the progression of important diseases [11]. Peroxide is involved in induction of oxidative stress and has also been linked to a variety of ailments such as inflammation, rheumatoid arthritis, diabetes, and cancer in humans [12]. Atomic absorption spectrometry (AAS), Inductively coupled plasma-mass spectrometry (ICPMS) and ion chromatography are the conventional analytical methods for the determination of metal ions [13,14] while titrimetry [15], fluorescence [16], chemiluminescence [17], and spectrophotometry [18] are used for H2O2 analysis (Table S2). Nevertheless, these analytical methods have good accuracy and reproducibility, but they involve sophisticated instruments,

https://doi.org/10.1016/j.mset.2019.08.001 2589-2991/Ó 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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operated by skilled and experienced technicians with more time consuming and reagent consumption along with labelling that leads to increases in the cost of analysis [19]. Therefore, development of easy-to-use, label free and more cost-effective methods is required for regular onsite monitoring of these pollutants. Electrochemical biosensor is the substitute for the traditional analytical methods and offers low cost, less sample pre-treatment and higher sensitivity with less time of analysis [20,21,22,23]. Electrochemical biosensors have also been utilized in detection of various biomolecules for disease diagnosis [24–28]. Some metal ions act as enzyme inhibitors, consequently decline activity of enzyme below the usual value that work as a biomarker of possible metal poisoning. The enzyme inhibition methods expand the potential applications of biosensors and offers substitute methods for determination of heavy metal ion like chromium, lead, mercury [29–31] and arsenic traces [32]. Enzymes including urease [30], acetylcholine esterase [32], and glucose oxidase [29]; tyrosinase [31] and horseradish peroxidase [33,34] have widely been used for the construction of this type of biosensor. Performance of peroxide based electrochemical sensor depends on the physiochemical property of modified electrode films, enzyme immobilization techniques involved such as cross-linking [35], adsorption [36], layerby-layer assembly [37], surfactant-enzyme complex formation [38] and functionalized nanomaterials used. Graphene, a two dimensional carbon-based nanomaterial has attracted significant research interest because of its large specific surface area, mechanical, thermal, optical and high electrical conductivities properties [39,40]. Besides graphene has good electrocatalytic activity which proves it as a perfect material for the fabrication of electrochemical nano-biosensors [41–43]. For enhancing the sensitivity of electrochemical sensors electron transfer mediators like different organic dyes such as methylene

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blue [44], prussian blue [45] and thionine [46] are generally employed. The thionine functionalized graphene nanocomposites has widely been applied for biochemical sensing in recent years [47–50]. Thionine (Lauth’s violet) belongs to the family of thiazine dyes and particularly attractive among the mediators. Thionine provides a highly conductive favorable sensing platform for successful immobilization of HRP enzyme on thionine layer using the glutaraldehyde. Amine group on thionine provide ease to conjugate surface for attachment with carboxyl terminal of HRP. In the present work, we fabricate the electrode with electroreduced graphene oxide layer deposited with electropolymerized thionine film. Horseradish peroxidase (HRP) enzyme is immobilized on fabricated electrode for the electrochemical based for sequential determination of H2O2 and indirect inhibitive measurement of Cr (VI). Voltammetry techniques are used here to obtain signal responses from the redox potential and current behaviour at the interface of electrode and H2O2 solution. Cyclic and linear sweep voltammetry showed an increase in the reduction current due to the electrocatalytic reduction of peroxide. Further reduction current is exploited for determination of chromium (VI) through inhibitive measurement (see Scheme 1). Decrease in the reduction current was observed with increase concentration of chromium due to inhibition of HRP enzyme activity. The approach of inhibition was studied using the Dixon and Cornish-Bowden plots.

2. Experimental 2.1. Chemicals and materials All reagents were of analytical grade and were used without further purification. Phosphate buffer solution (0.1 M, pH 6.5) pre-

Scheme 1. Stepwise fabrication process for the development of graphene-based biosensor for sequential determination of H2O2 and inhibitive measurement of Cr(VI) through cyclic voltammetry.

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pared from di-sodium hydrogen phosphate and di-hydrogen sodium phosphate was used as supporting electrolyte for all the electrochemical measurements. Thionine acetate, HRP and bovine serum albumin were purchased from Sigma Aldrich. Graphite powder with 7–11 mm was procured from Alfa Aesar for the preparation of graphene oxide. Hydrogen peroxide 30% (w/w), gluteraldehyde (25%) and all others reagent was purchased from Merck. All solutions were prepared using Milli-Q water (resistivity 18 MXcm at 25 °C). 2.2. Instrumentation Voltammetry experiments have been performed using a high resolution galvanostat/potentiostat based electrochemical workstation from Metrohm Autolab PGSTAT 101. A conventional three electrode based electrochemical cell set up with working electrode as glassy carbon electrode (GCE, U = 3 mm) and platinum wire as counter electrode were used. All potentials were measured using Ag/AgCl as a reference electrode. The NOVA software of version 1.10 was used to perform with computer to control all the input parameters and analysis of output signal response. All the electrochemical experiments were carried out at room temperature (25 ± 2 °C). UV–VIS spectrophotometry was done by Perkin Elmer. The X-ray diffraction (XRD) patterns were recorded using Rigaku, SmartLab with Cu Ka radiation (k = 0.15406 nm). The operating voltage and current was 40 kV and 30 mA respectively. Scanning electron micrographs were obtained with a ZEISS EVO15 scanning electron microscope (Japan) at an acceleration voltage of 15 kV. Atomic force microscopy characterization has been done by Agilent 5500 and ITO coated glass plate utilized as substrate for AFM characterization. The experimental conditions for the ITO coated film were done with similar conditions of GCE electrode for AFM analysis. The pH measurements were carried out with a Euteck pH-meter. 2.3. Fabrication of the sensor platform 2.3.1. Electrode pretreatment Prior to deposit graphene layer, the GCE was polished with 0.3 lM and 0.05 lM a-Al2O3 powder until a mirror-shiny surface was obtained, and it was then sequentially ultrasonicated in ethanol and millli-Q water for 15 min respectively. Subsequently the bare GCE were pre-treated by potential cycling from
1.0 to +1.0 V vs reference electrode at a scan rate of 100 mV s
1 in 0.1 M H2SO4 solution, in order to decrease the background currents and reproducible electrode response. 2.3.2. Preparation of GO and electroreduced graphene oxide layer Graphene oxide (GO) was prepared from graphite powder by using modified Hummers method. Briefly, graphite flake (2 g) and NaNO3 (0.50 g) was added to concentrated H2SO4 (100 ml) and was stirred in an ice bath for 30 min at controllable temperature of 1 °C. KMnO4 was added slowly into the mixture, and the temperature was maintained at 12 °C. The mixture was then transferred to a 35 °C water bath and stirred for 3 h. Then water was added consequently to produce a large exothermic reaction with 96 °C. External heating was introduced to maintain the reaction temperature at 96 °C for 90 min. Afterward, extra water (300 ml) was added, followed by the slow addition of 30 ml H2O2 (30%). Finally, the reaction mixture was cooled and washed several times with hydrochloric acid and distilled water using a centrifuge. Then the resulting GO solution was filtered and dried in a vacuum oven. Synthesized GO (1 mg/ml) was mixed with water to produce a yellowbrown suspension. Sensor platform was developed by coating GO suspension of 10 mL aliquot on the surface of GCE using drop cast method and dried in a vacuum desiccator denoted as GO/GCE electrode. Electroreduced graphene oxide (ERGO) was prepared using

electrochemical reduction method by applying cyclic voltammetry. The electrochemical reduction of GO was carried out by applying negative potential as discussed elsewhere [51–54]. Briefly, electrochemical reduction was done by scanning in a potential range between 0 and
1.8 V vs Ag/AgCl in PBS at 6.5 pH for 15 cycles at 100 mV s
1 and electrode is denoted as ERGO/GCE. 2.3.3. Electro-deposition of thionine on ERGO/GCE Electro-deposition of thionine has been carried out by applying a two-step process. Firstly thionine deposition was carried out through chronoamperometry at a constant potential (
1.5 V) for 300 s. After applying constant potential the growth of thionine film was carried out by cyclic voltammetry scan of 25 cycles from
0.3 to 0.6 V at 100 mV s
1 containing 1 mM thionine solution in 0.1 M phosphate buffer at pH 6.5 to obtain the TH/ERGO/GCE. 2.3.4. HRP immobilization (HRP/TH/ERGO/GCE) HRP was immobilized onto the TH/ERGO/GCE layer by crosslinking with glutaraldehyde and bovine serum albumin (BSA) so as to sustain the enzyme closer to its natural environment [55]. Briefly, A mixture of 35 mL of (0.5 mg/ml dissolved in 0.1 M PBS of pH 7.0) HRP solution, 10 mL of 1% BSA and 6 mL of 0.5% glutaraldehyde was prepared; 10 mL of this mixture was dropped onto the surface of the TH/ERGO/GCE electrode and allowed to dry for 2 h at room temperature. The electrode was enclosed with a beaker so that water can evaporate slowly and a uniform film can be formed. The resulting cross-linked enzyme electrode was marked as HRP/TH/ERGO/GCE and was stored with phosphate buffer solution at 4 °C in a refrigerator when not in use. 2.4. Response measurement The electrochemical response measurements were performed in an electrochemical cell through cyclic voltammetry and linear sweep voltammetry techniques. There is increase in the reduction current for H2O2 response with HRP immobilized electrode. The detection principle of Cr(VI) used in this study was based on the inhibitory effect on HRP. The decrease in the reduction current obtained for H2O2 by HRP using linear sweep voltammetry was estimated for metal ion concentration. The complete procedure was carried out in a three step. The HRP/TH/ERGO/GCE electrode were immersed into a 0.1 M PBS (pH 7.0) and known concentration of H2O2 was added to record a steady-state reduction current (Ii). After taking response from H2O2 the electrode was incubated with known concentrations of Cr (VI) solution for 20 min. The response was again measured and this corresponded to steady-state current after inhibition (If). The percentage of HRP inhibition (I %) and percentage residual enzyme activity (REA %) were calculated using Eqs. (1) and (2) respectively [56,57]. The effects of pH and temperature for fabricated sensor have been illustrated in Supportive information (Figs. S2 and S3). The pH and temperature of buffer solution have been maintained at 7.0 and 25 °C for all the experiment.

ðI%Þ ¼

ðI i
I f Þ  100 Ii

ðREA%Þ ¼

ðI f Þ  100 Ii

ð1Þ

ð2Þ

3. Result and discussion 3.1. Characterization of graphene sheets Fig. 1A shows SEM images of GO flakes having sheets with edge of mm size and its morphology resembles thin curtain indicating

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Fig. 1. (A) SEM images for graphene oxide (B) XRD Spectra of graphite powder and graphene oxide nanomaterial. (C) UV–VIS spectra of the various combination of HRP, GO and thionine.

very good exfoliation of graphite during the oxidation process. The XRD patterns of pristine graphite and synthesized GO sheets are presented in Fig. 1B. The data was collected between scattering angles (2h) of 5–70 at a scanning rate of 1.2° min
1. Pristine graphite shows the sharp diffraction peak at 26.48° (0 0 2) whereas the diffraction peak of GO appears at 10.58° (0 0 2), which is the characteristic peak of GO. The disappearance of the peak at 26.48° suggests the formation of GO during the oxidation process [58].

is slight decrease in the peak of enzyme mixed thionine and GO composite near 402 nm. The decrease in absorbance peak (curvee) might be due to adsorption and surface interaction phenomena. Both the spectra of GO and thionine not have any absorption peak near the HRP wavelength band. Therefore the wavelength band of HRP/TH/GO near 402 nm is merely due to the HRP Soret band. It is further concluded that HRP structure was almost remained unaffected in GO and thionine composite film. 3.3. Fabrication of modified electrode

3.2. UV–VIS spectrometry characterization of HRP/GO/thionine composite Absorbance spectrometry was conducted to explore interactions between GO and thionine. Also these absorption measurements were employed to confirm the stability of HRP in the composite film. Fig. 1C explains the absorption spectra of GO, thionine, HRP and thionine-GO suspension in water. The graphene oxide bands shows a strong absorption peak at 230.5 nm owing to p–p* transitions of aromatic double bond carbon (curve-a). Also a small bulge near 290 nm has been observed which corresponds to the n–p* transition of the C–O bond. Pure thionine spectrum shows two sharp absorption peaks at 282 nm and 597 nm for UV and visible region spectra respectively (curve-b). The decrease and peak shift in the absorption spectra of GO mixed thionine solution (curve-d) confirms the presence of thionine molecule immobilized with GO. The red shift near the visible absorption band and slight blue shift for UV band are found for GO mixed thionine composite was concurrent with previous reported work [47,48]. Blank HRP spectra found at 402 nm confirms its Soret band behaviour, whereas there

3.3.1. Electro reduction of graphene oxide Electrochemical reduction of GO is a nontoxic approach for the synthesis of reduced GO as compared to chemical reduction method [51,59]. The electroreduced GO coated electrode surface is irreversible, insoluble and stable in aqueous solution. Fig. 2A showed the electro-reduction process of GO film through CVs. Results showed characteristic enhanced reduction peak current at
1.43 V indicating strong reduction of GO observed under first cycle of CVs. This large cathodic peak response is resulted from reduction of the surface oxygen groups on GO. On further scanning, the cathodic peak drastically decreased and almost disappeared, revealing irreversibly reduction of surface-oxygenated group on graphene surface. The fabricated ERGO layer on GCE not only enhance the redox behaviour but also provided large surface area for grafting thionine molecules. The ERGO modified GCE electrodes showed enhanced electrochemical window resulting in increased conductivity of graphene. Fig. 2B shows the typical cyclic voltammograms (CVs) response of GO and ERGO on GCE. The reversible and stable peaks were observed for ERGO/GCE with anodic and

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Fig. 2. (A) CV of Electroreduction of graphene oxide for fabrication of ERGO/GCE electrode in PBS for (pH 6.5) at a scan rate of 100 mV S
1. (B) Response of electroreduced graphene (ERGO) oxide with graphene oxide modified GCE electrode using cyclic voltammetry scan in PBS (pH 6.5).

cathodic peak potential of 129 mV and
75.6 mV (vs. Ag/AgCl electrodes) respectively. The ERGO film showed the much higher increase the current response with respect to graphene oxide film with peak separation of 205 mV (See Supplementary data). It is due to increase in conductivity of film by electrochemical reduction of graphene oxide. The peak separation is increased about 20 mV during the complete cyclic growth of thionine layer over ERGO/GCE layer. The thionine layer attached firmly over the ERGO film in GCE is smooth and permanent. The two amine functional groups presents in thionine have good interaction towards GO layer with p–p stacking force and might responsible for cross-linking.

2.89 nm as compared to ERGO surface (Ra = 1.15 nm). This is attributed to more cross linkage of thionine polymerized film. The small lumps in the entire region of the Thionine/ ERGO film are occupied by HRP in HRP/Thionine/ERGO surface. This confirms that the HRP is well immobilized in thionine/ERGO surface. Also the roughness profiles of HRP immobilized layer are higher than the others modified surfaces (Ra = 7.37 nm). Such rough surface is more suitable for the analyte penetration. The roughness parameter of all the modified films has been tabulated in Table S1.

3.3.2. Electro-deposition of thionine film Thionine are widely known for biological staining due to its planar aromatic structure. Due to synergistic non-covalent chargetransfer and p–p stacking force for conjugated p bonds of thionine molecule, it get easily adsorb on graphene oxide [50,54]. Thionine and graphene oxide matrix is found to be good mediator to facilitate the electron transfer between immobilized enzymes to the functionalized electrode system. Fig. S1 shows the comparable growth thionine film on two different modified GCE electrodes. ERGO/GCE electrode (curve-a) shows the growth at higher current peak than the GO/GCE (curve-b) electrode. The growth of thionine film over the ERGO/GCE layer has more significant redox growth with larger current response as compared with the growth in bare GCE electrode.

Fig. 4 shows the typical cyclic voltammograms of modified electrodes obtained at different steps of electrode fabrication. Bare GCE (curve-a) principally exhibits not well voltammetric response in the applied potential range of
0.8 to 0.8 V in 0.1 M PBS solution of pH 7. The GO layers attached with GCE have shown increased reduction current response as compared to bare GCE electrode. Electro-reduction of GO (i.e. ERGO/GCE) results in the enhanced electrochemical redox window showing anodic and cathodic peak potential at 0.15 V and
0.1 V respectively. This is due to more C/O ratio and larger electroactive sites presents in reduced graphene oxide film. Electrodeposited thionine film (curve-d) show a pair of well-defined redox peaks with anodic (Epa) and cathodic potential (Epc) of
102.5 mV and
210 mV, respectively at a scan rate of 100 mVs
1 with peak separation (DEp) of 53 mV that is attributed to the redox reaction of immobilized thionine layer indicating the good interaction between ERGO and thionine molecule facilitate the electron transfer from thionine toward electrode surface. The ERGO surface allows more electron hopping process between adjacent redox groups. The formal potential of
0.155 V is attributed for both oxidation and reduction of thionine. The electrodeposited thionine film over the ERGO layer have more enhanced reversible responses, as compared with those of other thionine functionalized layer [60,61]. After the immobilization of HRP over the thionine film, the intensity of redox responses are decreased. The cathodic peak potential is significantly shifted to more negative potential of
0.527 V whereas the anodic peak potential has been observed at
0.146 V. The formal potential of HRP immobilized electrode has been shifted negatively more than two fold comparing with TH/ERGO/GCE electrodes. This confirms the well immobilization of HRP over the thionine modified ERGO platform and agrees with previous reports [62,63]

3.4. AFM characterization of ERGO, ERGO/Thionine and ERGO/ Thionine/HRP film The surface topography of ERGO, Thionine/ ERGO and HRP/ Thionine/ERGO film were characterized by AFM. All the recordings of AFM images are taken at ambient conditions in non-contact mode. Fig. 3(A) shows the AFM images with scan size of 1 mm2 area of electroreduced graphene oxide layer in three-dimensional topography. ERGO film shows more aggregated lumpy and wrinkled surfaces. This is attributed to increase in van der Waals force between adjacent layers due to the removal of oxygen functional groups during electrochemical reduction. The ERGO film observed as submicro islands due to increased p-interaction and hydrophobicity. The AFM images of Thionine/ ERGO surface and HRP/ Thionine/ERGO, layers are shown in Fig. 3(B) and (C) respectively. Thionine/ ERGO surface has average surface roughness (Ra) of

3.5. Electrochemical characterization of working electrode

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Fig. 4. Response of different modified electrodes at a scan rate of 0.1 V in 0.1 M PBS (pH 7).

R2 = 0.985 and Ipc (mA) =
25.9
740.4 m (V/ s), R2 = 0.965 for anodic and cathodic current respectively confirming surfacecontrolled electrode process. The surface concentration of electroactive species can be predicted from the slope of the plot of peak currents vs scan rate in accordance with the Brown Anson model (Eq. (3)) [64–66]

  Ip ¼ n2 F2 ACm =4RT

ð3Þ

where n is the number of electrons transferred, F is the Faraday constant (96487 C/ mol), A is the surface area of the electrode (0.0707 cm2), C is the surface concentration, m is the scan rate (V/ s), R is the gas constant (8.314 J/mol.K) and T is the absolute temperature of the system (293 K). The average surface concentration of thionine was estimated to be 1.43  10
9 mol/cm2 indicating very high packing of thionine molecule over ERGO layered GCE electrode. The charge transfer coefficient (a) and electron transfer rate constant (Ks) can be obtained by Laviron’s theory and Eq. (4) for the surface-confined electroactive species. For this purpose the anodic and cathodic over-potential (DE = Ep
E0) variations were studied with the logarithm of scan rates [65]. Fig. 5C shows values of DE proportional to the logarithm of the scan rate with regression equation of Epc (log m) =
0.106(V)
0.149 and correlation coefficient of 0.995 at the scan rates of 125 mV s
1. The slope of the DEcathodic vs. log (m) was approximately 106.1 mV and the charge transfer coefficient, a = 0.548 was obtained. Fig. 3. AFM 3D topography of (A) ERGO (B) Thionine/ERGO (C). HRP/Thionine/ERGO surface at scan size of 1 mm2.

3.5.1. Redox behaviour with scan rate To confirm surface confined current response of thionine/ERGO/ GCE fabricated electrode, we examined peak currents response with scan rate in 0.1 M phosphate buffer solution (PBS) (pH 7.0) (see Fig. 5A). The cyclic voltammetry of TH/ERGO/GCE gave almost symmetric anodic and cathodic peaks on increasing with scan rate. The anodic peak potential has been shifted towards positive potential whereas the cathodic peak potential moved towards more negative potential direction. The peak separation of the redox was increased about 10 fold on increasing the scan rate from 10 mV s
1 to 450 mV s
1 with no change in formal potential (154 mV) indicating the good redox reversibility of modified film. Fig. 5B. shows the linear dependence of the redox current response with scan rate up to 0.25 V/s in 0.1 M PBS with Ipa (mA) = 16.9 + 967.2 m (V/s),

Log ¼ alogð1aÞ þ ð1aÞlogalogðRTÞlogðRT=nFmÞað1aÞðnFDE=2:3RTÞ ð4Þ Using the charge transfer coefficient a of 0.548 and a scan rate of 100 mV s
1 the electron transfer rate constant (Ks) for TH/ERGO/ GCE electrodes was estimated to be 9.96 s
1. 3.6. Determination of H2O2 using HRP immobilized TH/ERGO/GCE electrode To cover the analytical relevance of the modified electrode, electrocatalytic activity of the TH/ERGO/GCE electrode towards the reduction of hydrogen peroxide was studied (Fig. 6). It is found that HRP immobilized over the electrodeposited thionine layered with electroreduced graphene oxide film has good electrocatalytic behaviour. On adding firstly 0.03 mM H2O2, cathodic peak potential was considerably increased to 26 mV. The catalytic current increased linearly with the increasing concentration of H2O2.

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Fig. 5. (A) Cyclic voltammograms of the TH/ERGO/GCE electrode at different scan rates from inner to outer (a) 10, (b) 30, (c) 50, (d) 75, (e) 100, (f) 125, (g) 150, (h) 175, (i) 200, (j) 250, (k)300, (l) 400, (m) 450 mV s
1 in PBS (pH 7.0) (B) Plot of cathodic and anodic peak current with scan rate (C) Plot of overpotential (DE) with logarithmic of scan rate.

Fig. 6. Cathodic sweep of cyclic voltammogram for the HRP/TH/ERGO/GCE electrode in PBS pH 7.0 with increasing concentration of H2O2 from (a–f; 0, 0.03, 0.08, 0.21, 0.3, 0.35 mM) at 100 mV s
1. Inset figure showing the calibration plot for peak reduction current with concentration.

Linear least square calibration graph over the range is shown in the inset of Fig. 6. The current response was proportional to the H2O2 concentration in the applied range from 0.03 mM to 0.35 mM with the linear regression equation (Ip =
6.983 C
28.10; R2 = 0.975). The detection limit was estimated to be 4.65 mM at a signal-tonoise ratio of 3. The regression line fits well with the experimental

data and can be applied in the unknown sample analysis of H2O2. Further, the comparison of developed fabricated sensor (HRP/TH/ ERGO/GCE) with existing biosensor is shown in Table S2. The developed highly uniform and stable layer in fabricated sensors provides good reproducible response towards H2O2 with comparable detection limit.

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3.7. Application of HRP/TH/ERGO/GCE electrode for Cr (VI) inhibition The potential mechanism of immobilized HRP on an electrode for electrocatalytic activity can be expressed as follows [67,68].

H2 O2 þ HRPred ! H2 O þ HRPox HRPox þ THred ! HRPred þ THox THox þ 2Hþ þ 2e ! THred Net reaction : THox þ 2Hþ þ 2e ! 2H2 O The immobilized HRP act as enzymatic catalyst to reduce the H2O2 and further regenerated with the support of thionine redox mediator. Once an inhibitor is introduced in the electrolytic solution it is also attached with the active binding site of HRP and thus renders the above reaction causing decrease in the reduction current, which is directly proportional to the concentration and time of exposure of the inhibitor [34,69]. 3.7.1. Effect of incubation time The degree of inhibition depends on the pre-incubation time. The rate of enzyme inhibition by heavy metal ions was quite slow therefore evaluation of the pre-incubation time is very important for off-time measurements and on-site analyses [52]. In an attempt to optimize the incubation time on the response of the developed biosensor, the current response in 0.5 mg/L Cr (VI) at different incubation time in the test solution was examined. The percentage inhibitions with different incubation times were calculated using Eq. (1) and the results are depicted in Fig. 7A. From Fig. 7A it is observed that an increase in the percentage inhibition caused by an increase in incubation time whereas a reverse behaviour of decrease in residual enzyme activity was found with incubation time. It is elucidated that on increasing the incubation time more interaction between the Cr (VI) inhibitor and active site of the

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enzyme occurred. After 20 min, there is saturation in the current response indicating that all the active site of the enzyme are well interacted and very less active site exists. In this study, an incubation time of 20 min was chosen so as to obtain a low detection limit for further experiments. 3.7.2. Measurement of Cr(VI) ions The inhibitive measurement of concentration dependent Cr(VI) in 0.12 mM H2O2 is depicted in Fig. 7B. The percentage inhibition and residual enzyme activity with different Cr(VI) concentration is obtained under the optimized condition. The percentage inhibition increased with increase in concentrations and found to be linear in the range of 0.039–0.78 mM with a regression equation of Percentage Inhibition (%) = 4.41(±1.24) C (mM) + 6.47(±0.59) with correlation coefficient of 0.989 (n = 3). The inhibition sensitivity obtained with the HRP/TH/ERGO/GCE electrode is 4.41% Inhibition mM
1 and the limit of detection is 0.02 mM (S/N = 3). The obtain value is much lower than the previously obtained from glucose oxidase based biosensor of 48 mM and 1.6 mM for HRP immobilized on Poly neutral red based carbon film electrode [70,71]. 3.7.3. Investigation of the inhibition mechanism and kinetics Enzyme has active site for substrate binding and when the substrate binds with enzyme active site it gets conformational changed to form the resultant product. The mechanism of enzymatic reversible inhibition depends on types of inhibitor and substrate. It may be competitive, non-competitive, and uncompetitive or mixed inhibition [72,73]. In this study, increasing concentrations of the trace metals and of the substrate, H2O2 was utilized to study the type of inhibition shown by Cr(VI) over immobilized HRP. To explore the type of enzyme inhibition, Dixon plots and Cornish-Bowden plots were utilized simultaneously. The Dixon plot is representation of the inverse of the enzyme activity vs inhibitor concentration plotted in Fig. 8A. The ratio of H2O2 concentration and enzyme activity is plotted vs inhibitor concentration in Cornish-Bowden plot as depicted in Fig. 8B. Both these plots were made for two different H2O2 concentrations, 0.15 and 0.45 mM. In Dixon plot, the two lines are parallel to each other inferring of uncompetitive inhibition. This is again confirmed by intersection of the two lines in second quadrant above inhibitor axis of Cornish Bowdon plot. The dissociation constant (K0i ) of the substrate enzyme-inhibitor-complex, was determined as 5.5 mM. Uncompetitive inhibition requires that one or more substrates bind to enzyme before the inhibitor can bind. In the present work uncompetitive inhibition mechanism is observed where the HRP enzyme and inhibitor Cr(VI) binds only to the complex formed between the enzyme and the substrate H2O2 (Fig. 8). The resultant (E-I-S) enzyme-inhibitor-substrate complex only undergoes reaction to form the product slowly. Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme-substrate complex and not free enzyme. 3.8. Stability, repeatability and restoration

Fig. 7. (A) Inhibitive response of 0.5 mg L
1 Cr (VI) with incubation time in 0.1 M PBS (pH 7.0) and 0.12 mM H2O2. (B) Concentration dependent response of enzyme inhibition and percentage residual enzyme activity with Cr(VI) towards H2O2 reduction.

The practical ability of enzyme based sensor is determined by the studying the stability factor for the electrode. Stability of the sensor was investigated and no significant decrease in biosensor response after 10 successive H2O2 measurements was found. The response of the sensor measured after two weeks stored in 0.1 M PBS pH 7 at 4 °C was decreased about 2.89%. The percentage stability of modified electrode in detection of H2O2 is 98% after measuring with 50 cyclic scan (see Supplementary information Fig. S4). After 10 successive measurement of chromium, the enzyme lost its activity with 3.58%. The repeatability of the HRP/TH/ERGO/ GCE electrode was investigated for fixed Cr (VI) concentrations. A

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Fig. 8. (A) Dixon plot (B) Cornish plot (C) Schematic representing enzyme inhibition mechanism for biosensor response.

relative standard deviation (RSD) of 3.78% was calculated based on five successive measurements. The immobilized HRP activity after inhibition was not restored properly with buffer washing. Lost enzyme activity was restored on incubation with 10 mM EDTA in PBS buffer for 2 h, 90% of the lost enzyme activity was restored.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

4. Conclusion

Acknowledgments

In this paper, we have described enzymatic H2O2 biosensor based on electrodeposition of thionine film over electrochemically reduced graphene oxide layer. In the fabricated matrix, enzyme is immobilized by cross-linking with glutaraldehyde and BSA to prevent the leakage of enzyme and maintain its natural environment. These sensors showed the more stable and reproducible response for H2O2. This biosensor also applied for detection of Cr(VI) by studying the inhibitive effect on the HRP enzyme activity. The sensitivity of H2O2 with the HRP/TH/ERGO/GCE electrode based biosensor was 6.98 mA/mM whereas for Cr(VI) the limit of detection found to be 0.02 mM. Enzyme inhibition reaction is not a selective process, so the method could be employed only for the measurement of overall inhibition of enzyme by the total amount of heavy metals in water sample. Limited selectivity of the method could be overcome by coupling a chromatographic based separation technique with the amperometric measurements. The inhibition mechanism determined from Dixon and Cornish-Bowden plots was found to be uncompetitive with dissociation constant (K0i ) of 5.5 mM. Developed sensor showed good stability and reproducibility that could be explored for environmental analyses and clinical testing. In spite of obtaining prominent achievements, the complicated procedures and inconvenience in the fabrication of platform apparently hindered their sensing applications. Therefore, new fabrication technologies are anticipated in the future in response to these prospects. The future aspects of this work are in designing and development of low cost microchip analyser. The flexible plastic based substrate may be anticipated in coming future for regular on field monitoring of H2O2, chromium and other metal ions similar with available commercially reliable glucometers.

S. K. Singh sincerely thank the Department of Science and Technology (DST)-Nanomission [DST/NM/NB/2018/40 (G)] and DSTINSPIRE (IFA12-LSBM-31), Government of India for the financial support. The authors also would like to acknowledge TEQIP-II, Motilal Nehru National Institute of technology (MNNIT) Allahabad India for the research support. The authors are thankful to the Director, Motilal Nehru National Institute of Technology Allahabad for providing the Centre for Interdisciplinary Research (CIR) experimental facilities.

Declaration of Competing Interest

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