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Highly Redox Active Hematin-Functionalized Carbon Mesoporous Nanomaterial for Electro-Catalytic Reduction Applications in Neutral Media Khairunnisa Amreen, and Annamalai Senthil Kumar ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00337 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018
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Highly Redox Active Hematin-Functionalized Carbon Mesoporous Nanomaterial for ElectroCatalytic Reduction Applications in Neutral Media
Khairunnisa Amreena and Annamalai Senthil Kumara,b,c* a
Nano and Bioelectrochemistry Research Laboratory, Department of Chemistry,
School of Advanced Sciences, Vellore Institute of Technology University, Vellore-632014, India b
Carbon dioxide Research and Green Technology Centre, Vellore Institute of Technology University, Vellore-632014, India c
Institute of Biochemical and Biomedical Engineering, National Taipei University of Technology, Taipei 10608, Taiwan (R.O.C.)
Keywords: Hematin; Redox Function; Surface-Confinement; Graphitized Mesoporous Carbon; Chitosan; Chemically Modified Electrode; Electrocatalytic Reduction Reactions.
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ABSTRACT. Hematin is a hydroxyl group linked heme site (hydroxyl heme) of the natural enzymes/proteins like hemoglobin, cytochrome c, catalase and horseradish peroxidase and it has an important role in the physiological function. Owing to the problems like poor electron-transfer functionality (on solid electrodes), less-solubility and molecular aggregation in aqueous solution, limited electrochemical studies were reported in the literature. A new electrode modification method for hematin using graphitized mesoporous carbon nanomaterial and chitosan for enhanced redox active and efficient electro-catalytic reductions of hydrogen peroxide and dissolved oxygen in neutral pH have been demonstrated in this work. The hematinmodified electrode showed a highly stable redox peak at Eo’=-0.390 V vs Ag/AgCl with heterogeneous rate constant value of 1.34 s-1. Calculated hematin-active loading concentration (Γhemat = 196×10-10 mol cm-2) is about twenty times higher than the literature reported values. Physicochemical and electrochemical characterizations revealed trapping of the Hematin via axial bond coordination and intermolecular-hydrogen bonding with amino functional groups of chiotsan and π-π interactions with the graphitic site of mesoporous carbon within the matrix. The new hematin electrode showed about 400 mV reduction in the over-potential with current sensitivity/detection range of 570 nA µM-1/100-900µM and 6.7µA ppm-1/1-10 ppm respectively for H2O2 and dissolved oxygen reduction reactions in pH 7 phosphate buffer solution. MichaelisMenten kinetics were applied for the H2O2 reduction reaction and estimated the rate constant values as KM =0.78 mM and ks = 1.15s-1. No marked interference was noticed with common biochemicals such as nitrite, nitrate, glucose, uric acid, ascorbic acid, xanthine, hypoxanthine, cysteine and dopamine on amperometric i-t detection of H2O2 indicating the activity similar to the heme based proteins/enzymes.
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1. INTRODUCTION Inspired by nature, mimicking the intricacy of naturally occurring enzymes has been a long-term goal of the researchers. Biomimetic electrocatalytic reductions of O2 and H2O2 are of great importance in the fields of electrochemistry1, electroanalytical chemistry2, bio-medical3, energy4,5 and food science industry6,7. In this connection, synthetic bio-inspired molecules reduce the gap between the natural and artificial enzymes/proteins. 8-12 Hematin (Hemat), one of iron porphyrin derivatives, is a well-known active center of heme-proteins and it has the ability to mimic the active site of various heme-proteins/enzymes such as catalases, peroxidases, hemoglobin (Hb) and myoglobins. 13-17 Because it has Fe3+ as a central metal atom to which a hydroxyl (-OH) group is linked, in general, it is referred as oxyheme or hydroxyheme. One of the major functions of Hematin is that it triggers the production of globulin in the body.18 Indeed, direct application of Hematin is of significant challenge because of its poor solubility, molecular aggregation in aqueous solution and oxidative self-destruction during oxidation. In fact, these problems weaken the catalytic activity and limit its wide applications as an effective homogeneous catalyst. Herein, we report hematin stabilized graphitized-mesoporous carbon nanoparticle(GMC)/chitosan
(Chit)
modified
glassy
carbon
electrode,
designated
as
GCE/GMC@Hematin-Chit, as a perfect model for the peroxidase enzyme functional activity and for electrochemical applications like oxygen and hydrogen peroxide reduction reactions in neutral pH solution. Since 1998, there have been efforts to immobilize hematin on various modified electrode surfaces and to turn its electron-transfer function. In this regard, a spray-pyrolysis technique prepared carbon films19, histidine-micellar environment of sodium dodecyl sulphate20 and TiO2 nanoparticle printed indium-tin-oxide electrodes21 have been reported to stabilize the hematin.
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Although it was successful to immobilize, a specific redox peak at an apparent standard electrode potential, Eo’ = -0.380 V vs Ag/AgCl corresponding to the ET of hematin-Fe3+/2+-OH in neutral pH solution22, could not have been achieved. These observations support the difficulty in turning the ET of the hematin. In 2009, Ju group, first demonstrated the ET feature of the hematin by immobilizing it in a 1-butyl-3-methylimidazolium hexafluorophosphate ionic liquid-single walled carbon nanotube (SWCNT) suspension modified glassy carbon electrode. 23 The modified electrode showed a redox peak at Eo’ = -0.380±0.001 V vs Ag/AgCl and ΓHemat = 4.5×10-10 mol cm-2 in pH 7 phosphate buffer solution (PBS). Indeed, later attempts on naked SWCNT (Eo’=0.05 V vs Ag/AgCl; ΓHemat=10×10-10 mol cm-2) and carboxylic acid functionalized multiwalled carbon nanotube-chitosan- poly(acridine red) based composite (Eo’ = -0.272 V vs Ag/AgCl with ΓHemat=1.23×10-10 mol cm-2)24 resulted in partial fulfilment of the biomimitic characteristics. A positive Eo shift noticed in the above cases are due to unknown interfacial resistance values. In 2014, Yu et al have introduced hematin-poly(3,4-ethylenedioxythiophene) (PEDOT, electro-polymerization preparation)-1-pyrenebutanoic acid-ZrOCl2 based composite system, which is connected through pi-pi stacking, for the electron-transfer reaction (Eo’~-0.38 V vs Ag/AgCl; ΓHemat = 10.9×10-10 mol cm-2).25 In general, these kinds of preparations require laborious multiple-coatings and immersions procedures. Meanwhile, recently, a bicarbonate solution pre-anodized GCE that has specific pores on the surface, has been reported for hematin immobilization. 26 This system showed Eo’ = -0.3 V vs Ag/AgCl and ΓHemat = 0.9×10-10 mol cm-2. Interestingly, a new hematin immobilization method introduced in this work based on GMC and Chitosan which can be prepared within 15±1 minutes has shown excellent biomimicking redox signal at Eo’ = -0.380 V vs Ag/AgCl with the highest ΓHemat=126×10-10 mol cm-2 (active site loading) suitable to perform various electrocatalytic applications.
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To show the efficient applications of the present system, electro-catalytic reduction of hydrogen peroxide and dissolved oxygen have been chosen as model systems since these reactions have been mostly performed using hemoglobin, cytochrome c and heme based bioelectrodes at Eo’~ -0.38 V vs Ag/AgCl in neutral pH solution. 27,28 It is noteworthy that compared with covalently modification, pi-pi bonding associated preparation of heme-chemically modified electrode showed highly efficient ETs.29,30 For instance, Diaz-Ayala et al. reported a hemoglobin covalently attached poly-Lys-MWCNT system has failed to show any specific redox peak for the heme at Eo’~ -0.380 V vs Ag/AgCl.
29
Unlike for enzyme based electrodes, present bio-
mimicking system is found to be economic, quick in preparation, highly stable and improved electrochemical performance towards the electro-catalytic reactions. Note that, hematin can be entrapped on the pores of the carbon mesoporous (nanomaterial) system. We proved it by performing various physicochemical and electrochemical experiments including several controls based on various carbon nanomaterials. In fact, the porous/cavity present in GMC has an addition feature apart from the graphitic structure and oxygen functional groups, for the molecular structure incorporation, improved stability and electron-transfer function.
2. EXPERIMENTAL SECTION 2.1 Materials and Reagents. Lyophilized hematin from porcine (purity >98.2%), graphitized mesoporous carbon (purity >99.95%, 200/mV;
log (ks/s-1)= αlog(1-α) + (1-α)logα–log[(RT/{nF(v/V.s-1)})]–α(1-α)nF∆Ep/2.3RT --(3)
where, α=average transfer coefficient value (0.53) calculated using the expression, Sa/Sc=α/1-α. Note that the Laviron model equation is applicable only when the ∆Ep value is higher than 200 mV.41 Calculated ks value for GCE/GMC@Hemat-Chit is 1.34 s-1 (v=160 mV s-1; ∆Ep = 210 mV). Obtained ks value is comparable with the literature reports on the hemoglobin based surface-confined redox systems like Hb-MWCNT-Nf (1.25 s-1), Hb-f-CNT-CTAB-Nf (1.25 s-1), Hb-IL-MWCNT-CPE (0.84 s-1), Hb-IL-MWCNT-CPE (0.99 s-1).42-44 These results indicate the suitability of the present system to extend into biomimicking of the heme based protein/enzyme applications. The influence of solution pH on GCE/GMC@Hematin-Chit was investigated by CV technique at a pH range 2-11 at a fixed scan rate=50 mV s-1. As can be seen in Figure 2D, the redox peaks are found to shift systematically with the solution pH. Eo’ value obtained at each condition was plotted against solution pH value as in Figure 2E. A negative straight lines in the regions, pH 3-9 and pH 9-11 with slope values -32 and -68 mV pH-1 were noticed. The deviation in the lines obtained at pH~9 is due to pKa value of the hematin on the modified electrode
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surface. Note that at the pKa value, the hematin-composite chemically transformed to another protonated form. Due to this structural change, there is an alteration in the electron-transfer reaction and hence to variation in the slope values. The slope value obtained at about -60 mV pH-1 is considered as a Nernstian response with involvement of equal number of proton/electron in the electrochemical reaction. On the other hand, the slope value obtained at ~30 mV pH-1 is referred as a non-Nernstian response with involvement of unequal number of proton/electron as 1H+/2e- in the electrochemical reaction. In the literature, similar kind of non-Nernstian slope values were reported with Hb-polymer grafted MWCNT (−42 mV pH-1)
45
and Hb-CNT-HA
(−38 mV pH-1) systems. 46 Partial protonation of ligand is the likely reason for the observation. 28
At this stage, it is difficult to propose the possible interaction between the hematin with the
matrix systems. In further, effect of carbon surface-functional group on the hematin immobilization and several physicochemical characterization experiments were carried out to understand the structural feature of the Hematin modified electrode. 3.2. Effect of carbon on Hematin-CME. Figure 3 is a typical CV response of Hematin modified between carbon nanoparticles like mesoporous-hydrophillic (non-graphitized), mesoporous-hydrophobic (non-graphitized), carbon nanofibre (graphitized), carboxylic acid functionalized MWCNT, graphene oxide and graphite nanopowder and Chitosan layer as in Figure 3. With respect to the peak current, the order of electron-transfer feature of the hematin on the carbon nanomaterial/chitosan film is sequenced as follows (increasing order); CMhydrophobic
