An electrochemical biosensor developed for the online monitoring of H2O2 based on the reduced graphene oxide-cerium dioxide nanocomposite

Oct 16 th - 18 th 2019, Brno, Czech Republic, EU 396 AN ELECTROCHEMICAL BIOSENSOR DEVELOPED FOR THE ONLINE MONITORING OF H 2 O 2 BASED ON THE REDUCED GRAPHENE OXIDE-CERIUM DIOXIDE NANOCOMPOSITE 1,2 Atripan MUKHERJEE, 1,2,3 Amirmansoor ASHRAFI, 1,2 Zuzana BYTESNIKOVA, 1,2 Lukas RICHTERA, 1,2 Vojtech ADAM 1 Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic, EU, [email protected] 2 Central European Institute of Technology, Brno, University of Technology, Brno, Czech Republic, EU 3 CEITEC-Central European Institute of Technology, Mendel University in Brno, Brno, Czech Republic, EU Abstract A biosensor was developed for monitoring H 2 O 2 based on reduced Graphene Oxide-Cerium Oxide (rGO-CeO 2 ) nanocomposite (NC). The NC shows catalytic effect on the electrochemical reduction of H 2 O 2 at −0.3 V. The effect of the NC on the electrochemical reduction of H 2 O 2 was studied by cyclic voltammetry and compared with bare glassy carbon (GC) and GC/r-GO. Moreover, to improve the signal a complex containing copper was incorporated in the dispersion of the nanocomposite which acts as a mediator. It was found that the electrochemical reduction of H 2 O 2 takes place at less negative potential with increased peak current attitude at GC/r-GO-mediator. The developed sensor GC/r-GO-mediator, was applied for the continuous monitoring of H 2 O 2 by chronoamperometry. The applied potential was −0.3 V which results in the enhanced sensitivity of the developed biosensor. The developed biosensor shows good stability, and reproducibility. The reproducibility of the developed electrode was evaluated by calculation of relative standard deviation value 6.77%. The linear range was found to be 3.4 - 23.48 µg/ml. Furthermore, limit of detection and limit of quantification were 0.68 µg/ml and 2.27 µg/ml respectively. Keywords: Nanocomposite, reduced graphene oxide-cerium dioxide, biosensor, biomimetic, chronoamperometry 1. INTRODUCTION Hydrogen peroxide (H 2 O 2 ) a simple molecule in nature belongs to the class of reactive oxygen species with greater significance in the field of pharmaceutical, environmental, mining, textile, food manufacturing and processing industry, clinical and diagnostic field [1]. Physiologically and pathological importance of H 2 O 2 is associated with many important cellular process like signal transduction, immune cell activation, apoptosis, etc. [2,3]. H 2 O 2 is widely used in the food and processing industry as preservative due to its bactericidal activities and used as sterilization in the packaging industry [4]. However excessive use of H 2 O 2 in food packaging and processing is harmful for health as it acts as an adulterants leading to severe gastrointestinal problem [5]. In rainwater, H 2 O 2 can be formed due to photochemical reaction and present in industrial waste to contaminate the ground and surface water, a threat to the environment [6]. Therefore, continuous monitoring and sensitive detection of H 2 O 2 is important for environmental protection, food industry, clinical and diagnostics application [7-10]. In recent years, real-time monitoring of H 2 O 2 is significant to study the physiological and pathological changes leading to investigate the process of oxidative stress in vivo and in vitro due to its chemical properties. Several methods like spectrophotometric, fluorimetric methods were used for the detection, analysis and estimation of H 2 O 2 [11-13]. Several disadvantages like expensive chemical, require skilled technicians, extended time for the analysis and suffering from interferences are common issues with these conventional methods for continuous monitoring. Electrochemical detection is selective, sensitive, https://doi.org/10.37904/nanocon.2019.8613 Oct 16 th - 18 th 2019, Brno, Czech Republic, EU 397 simple, easy to use, accurate, cost-effective, and quick [6]. Moreover, amperometric methods can be applied to measure accurate kinetic information in real-time to image the continuous release of H 2 O 2 [14]. The detection and estimation of H 2 O 2 by using electrochemical amperometric techniques have attracted great attention for its application in biosensors and online monitoring. The development and preparation of sensors and biosensors were enriched after the discovery of the carbon nanomaterials. Different carbon nanocomposites (NC) such as single-walled carbon nanotubes, multiwalled-carbon nanotubes, graphene, graphene oxide (GO) and reduced graphene oxide (rGO) were used to promote and enhance electron transfer reaction in the development of biosensors [15,16]. Different types of sensors are used for the detection of biologically complex molecule like enzyme based biosensor, non-enzymatic sensors, nucleic acid based sensors, peptide based sensors. Enzymatic biosensors possess various challenges regarding the instability of the enzyme, immobilisation on the electrode surface leading to leakage and low effective binding on the surface of the electrode which causes low activity of the sensor. Moreover, enzymes increase the overall cost for the preparation of the sensor and put obstacles for the preparation of cost-effective sensors [17,18]. These serious challenges lead to the development of NC possessing biomimetic properties with stability, long lifetime, and cost-effective characteristics. For fast detection and successful analysis amperometric method will be relied on the applied potential. The applied potential was selected at less negative potential to acheive better sensitivity and stability of the developed sensor. The main aim of this work is to develop a non-enzymatic, cost effective, sensor for the rapid detection and analysis of H 2 O 2 with higher sensitivity, stability which is costeffective as well. A copper containing complex, tris(2,2’-bipyridyl)copper(II) dichloride was synthesized and modified accordingly to be used as a mediator together with rGO-CeO 2 to achieve sensitive analysis of H 2 O 2 at low potential [19,20]. 2. MATERIALS AND METHODS Graphite flakes (100 mesh, ≥75% min), Sulfuric acid (H 2 SO 4 ) , Potassium permanganate (KMnO 4 ) Nafion 117 (NF), Cerium trinitrate (Ce(NO 3 ) 3 ), Sodium chloride (NaCl), Potassium chloride (KCl), Sodium phosphate dibasic (Na 2 HPO 4 ), Potassium dihydrogen phosphate (KH 2 PO 4 ), N,N-Dimethylformamide (DMF), Hydrogen peroxide (H 2 O 2 ) are analytical grade and purchased from Sigma-Aldrich (St. Louis, MO, USA). The water (18.20 MΩ·cm) used for solution preparation was first double distilled by an Aqua Osmotic 02 (Aqua Osmotic, Tisnov, Czech Republic) and then deionized by using a Millipore RG (MilliQ water, Millipore Corp., Billerica, MA, USA). An Autolab electrochemical analyzer model PGSTAT-101 operated via NOVA 2.1 software (Metrohm Autolab, Utrecht, Netherland) was used for the voltammetry experiments. 0.01 M Phosphate buffer saline (PBS) was prepared with 0.132 M of NaCl, 0.0027 M KCl, 0.01 M of Na 2 HPO 4 and 0.0018 M of KH 2 PO 4 were mixed to prepare 1.0 l buffer solution. The pH was adjusted at 7.4 by 1.0 M NaOH. The entire study was conducted by using this buffer. The conventional three-electrode configuration was used in which a platinum electrode used as a counter electrode, Ag/AgCl 1.0 M KCl as the reference electrode and a modified glassy carbon electrode (GCE) served as the working electrode. 2.1 Preparation of nanocomposite (NC) Following Hummer’s procedure GO was prepared by the chemical oxidation of graphite flakes in a mixture of concentrated H 2 SO 4 and KMnO 4 [21]. The modified NC of rGO-CeO 2 was synthesized after getting inspired from the previous study [22]. Modified NC with mediator was prepared after mixing 1.0 ml of rGO-CeO 2 with 0.1 mg of copper mediator (M) tris(2,2’-bipyridyl)copper(II) dichloride sonicated for 40 min. 2.2 Preparation of modified electrode The bare GCE was polished on a polishing pad by using diamond suspension in water (0.5 micron) for 2 min followed by sonification in MilliQ water for 5 min and in absolute ethanol for another 5 min. The electrode cleaning procedure was repeated with polishing with alumina slurry of (1.0 micron) and (0.5 micron) Oct 16 th - 18 th 2019, Brno, Czech Republic, EU 398 respectively. The polished electrode was modified by using different composition of NC. Figure 1 presents modification of electrode by drop casting 10.0 µl of the NC on the bare electrode and allowed to be dried in room temperature (RT). 5.0 µl of 1.0% NF was drop casted and left for drying in RT. The NF was prepared by neutralization of NF using 8.0% ammonium solution. 0.1 M of H 2 O 2 was freshly prepared as stock solution as analyte. Figure 1 Preparation of modified electrode 2.3 Electrochemical measurement The electrochemical behaviour of H 2 O 2 at the developed electrode was studied by cyclic voltametry (CV). The CV parameters were start potential 0.0 V, stop potential +0.4 V, scan rate 50 mV/s, upper vertex potential +0.5 V and lower vertex potential −1.0 V. The potential was scanned towards anodic direction. The rapid electrochemical detection of analyte was conducted by chronoamperometry at the selected applied potential. The chronoamperometric study was carried at a selected potential of −0.3 V at working electrode for 3000 s. The current directed between the working and counter electrode was sampled every 1 s. 3. RESULTS AND DISCUSSION 3.1 Study of electrochemical behaviour of H 2 O 2 by CV The CV study was conducted with bare GCE, GCE modified with rGO, GCE modified with rGO-CeO 2 , GCE modified with rGO-CeO 2 and M as different NC in blank and 0.1 M H 2 O 2 . As seen in Figure 2 with addition of 250 µl of 0.1 M H 2 O 2 in PBS, a broad reduction current appeared at rGO. The attitude of the reduction increased at rGO-CeO 2 indicating an improved electro catalytic properties of the synthesized NC. However, including the M into the rGO-CeO 2 as rGO-CeO2+M resulted in a shift in the potential of the reduction peak to less negative potential and an increase in the reduction peak current. This might be due to the synergic electro catalytic and biomimetic properties of the NC along with the mediating activity that of the mediator. Figure 2 Electrochemical behaviour of different modified GCE using CV at 0.1 M H 2 O 2 Oct 16 th - 18 th 2019, Brno, Czech Republic, EU 399 As depicted in Figure 3 copper is first electrochemically reduced at the electrode surface from Cu(II) to Cu(I). Cu(I) in solution reduces the H 2 O 2 to water while itself oxidises to Cu(II) again. Therefore, the reduction current of Cu(II) to Cu(I) is related to the amount of the H 2 O 2 which reduces to water. The high surface, electro-catalytic and biomimetic properties of the NC results in an enhanced reduction current at such small potential magnitude. Figure 3 Expected mechanism of the NC 3.2 Amperometric study The sensitivity of the different developed electrodes was studied at an applied potential corresponding to the reduction of H 2 O 2 . For comparaing the sensitivity, different calibration curves obtained by the chronoamperometry at −0.3 V were plotted and the slopes of the calibration curves are represented in Table 1. As can be observed the highest sensitivity belongs to that of rGO-CeO 2 +M . Table 1 Detail sensitivity of different NC on GCE at −0.3 V Nanocomposites (NC) rGO rGO-CeO 2 rGO-CeO 2 +M Sensitivity (µA·ml/µg) 0.067 0.801 2.956 At −0.3 V, the sensitivity was found to be highest with GCE/rGO-CeO 2 +M/NF. The reproducibility of the sensor was studied by continuous injection of 20 µl of 0.1 M H 2 O 2 . All the experiments were carried out thrice to check the stability of the NC on the electrode surface along with the reproducibility of the sensor. Figure 4 Chronoamperometry and calibration curve of GCE/rGO-CeO 2 /NF at 0.01 M PBS The calibration curve of GCE/rGO-CeO 2 /NF and GCE/rGO-CeO 2 +M/NF with their corresponding chronoamperometric study were presented in Figure 4 and Figure 5 respectively. A good linearity between Oct 16 th - 18 th 2019, Brno, Czech Republic, EU 400 the reduction current and the continuous injection of H 2 O 2 in the reaction buffer was observed. The sensitivity of the GCE/rGO-CeO 2 +M/NF was found to be better in-comparison with the GCE/rGO-CeO 2 /NF electrode. Very low response was obtained with the GCE modified with GCE/rGO/NF. Figure 5 Chronoamperometry and calibration curve of GCE/rGO-CeO 2 +M/NF at 0.01 M PBS The limit of detection (LOD) and limit of quantification (LOQ) were calculated to be 0.68 µg/ml and 2.27 µg/ml with linear range of detection 3.4 - 23.48 µg/ml. The RSD% of the modified electrode was calculated as 6.77. 5. CONCLUSION The proposed study was conducted to develop a non-enzymatic sensor using modified carbon NC, able to detect H 2 O 2 with high sensitivity at small potential magnitute. The combining effect of the carbon NC with the copper mediator increases the electron transfer capacity and electrical conductivity to acts as a better transducer material. Increase in sensitivity, higher repeatability obtained with a simple, rapid and inexpensive method were the advantages of the prepared sensor. This preliminary study confirms the biomimetic property of the modified carbon NC to be used for the fabrication, modification, detection and analysis of analyte by the sensor. For future perspective this sensor can be modified into biosensor by immobilisation of enzyme which will be able to detect some complex biological molecule as analyte. Due to low applied potential, the stability and integrity of the enzyme will remain intact and expected to perform at highest activity. The selectivity and interference study will be performed in future to check the biomimetic activity of the modified carbon NC at different conditions. ACKNOWLEDGEMENTS The proposed study and research was financially supported by the Central European Institute of Technology at Brno. The copper mediator tris(2,2’-bipyridyl)copper(II) dichloride complex was kindly provided by Dr. Milan Sýs, Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Czech Republic. 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