Improved Electrical Wiring of Glucose Oxidase Enzyme with an In-Situ

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Improved Electrical Wiring of Glucose Oxidase Enzyme with an In-Situ Immobilized Mn(1,10 Phenanthroline)2Cl2-Complex/ MWCNT Modified Electrode Better than Os-Complex for HighCurrent Sensitivity Bioelectrocatalytic and Biofuel Cell Applications Natarajan Saravanan, Pinapeddavari Mayuri, and Annamalai Senthil Kumar ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00584 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on November 5, 2018

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Improved Electrical Wiring of Glucose Oxidase Enzyme with an In-Situ Immobilized Mn(1,10 Phenanthroline)2Cl2-Complex/MWCNT Modified Electrode Better than Os-Complex for High-Current Sensitivity Bioelectrocatalytic and Biofuel Cell Applications Natarajan Saravanan1, Pinapeddavari Mayuri,1 and Annamalai Senthil Kumar*1,2 1Nano

and Bioelectrochemistry Research Laboratory, Department of Chemistry, School of

Advanced Sciences, Vellore Institute of Technology University, Vellore-632 014, India 2Carbon

dioxide Research and Green Technology Centre, Vellore Institute of Technology University, Vellore-632 014, India

KEYWORDS. Electrical wiring; Glucose oxidase enzyme; Mn(phen)2Cl2 complex; In-situ surface confinement; MWCNT surface; Bio-electrocatalytic oxidation; Biofuel Cell.


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Abstract. Search for a new and efficient transducer that can electrically connect enzyme active site, like flavin adenine dinucleotide in the glucose oxidase (GOx), with electrode surface is

a

cutting-edge

research

work.

Currently,

Os(bpy)-complex

pendent

polyvinyl

pyridine/polyvinyl imidazole/pyridinium hydrogel based chemically modified electrodes have been

widely used for this purpose (bpy=2,2’ bipyridine). Herein, we report, a

[Mn2III(phen)4(O)(Cl)2]2+ multiwalled

carbon

complex/Nafion nanotube

MWCNT@Mn2(Phen)4O(Cl)2-Nf,

immobilized

modified phen=1,10

glassy

carboxylic carbon

Phenanthroline),

acid-functionalized electrode

prepared

electrochemical method using the precursor, Mn(phen)2Cl2, as an efficient,

by

(GCE/fan

in-situ

low cost and

alternate to the Os-complex transducer, for the Glucose oxidase enzyme (GOx) based bioelectrocatalytic system. The existence of the key active site, [Mn2III(phen)4(O)(Cl)2]2+ on the modified electrode was confirmed by physicochemical characterizations using Transmission electron-microscope, Raman, IR and UV-Vis spectroscopes and ESI-MS techniques. The Mncomplex modified electrode showed a redox peak at Eo’= 0.55 V vs Ag/AgCl in neutral solution with a surface excess (Mn) value, 5.6×10-9 mol cm-2. The GOx enzyme bioanode prepared by adsorbing GOx on the Mn-complex modified electrode has shown an efficient electrocatalytic oxidation of glucose with Tafel slope value 111 mV dec-1. Amperometric i-t analysis of glucose showed a calibration plot in a linear range of 50550 M and with current sensitivity 316.7 A mM-1 cm-2. The current sensitivity value obtained here is about 280,000 times higher than that of the Os(bpy)-complex based transducers used for the GOx based bio-electrocatalytic applications. Utilizing this new bioanode system along with a Pt-based oxygen reduction electrode, a new biofuel cell was constructed and achieved a power density value 7.5 W cm-2.



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1. INTRODUCTION Innovation in improving the molecular wiring and electron-transfer reaction between the enzyme’s redox active center, for instance flavin adenine dinucleotide (FAD) in the glucose oxidase (GOx) enzyme, and electrode-surface is a continued interest in the inter-disciplinary research areas of small molecular chemistry, bio-electrocatalytic and biosensor systems. Since, the redox active-site is deeply shielded by an insulated protein shell, in general, it is difficult task to shuttle the electron of enzyme active site with external source (solid electrode surface).1-3 To overcome this problem and to promote the flow of electrons, redox mediators, i.e., transducers or redox relays, like ferrocene and its derivatives,

4-7

quinones8,9, [Ru(bpy)3]2+ (bpy=2,2’

bipyridine)10 and Os(bpy) complexes pendent polypyridine, 1-3,11-16 have been coupled with the GOx enzyme system. Amongst them, with respect to the molecular wiring and current sensitivity, Os(bpy)2Cl2 crosslinked with cationic polymeric systems like polyvinyl pyridine (PVP) and poly(vinylimidazole) (PVI) have been considered to be the best and achieved a bioelectrocatalytic current signal in a window ~0.0270 A mM-1 cm-2.1-3,11-16 The sensitivity value has been further altered/improved by reducing the hydrophobic character (176.8 A mM-1 cm-2)17 and rigidity (0.004 A mM-1 cm-2)18, incorporation of gold nanoparticles and surfactant (28 A mM-1 cm-2)19,20 and functional groups like aldehyde and carbonyl (12.5 A mM-1 cm-2)21 in to the Os(bpy)-complex hydrogel system. Nevertheless, complicated preparation procedure of the redox mediator,14,18,20 high cost (OsCl62- is ~25 times higher cost than MnCl2) and hazardous nature of the precursor (oxidation of the Os-complex may reduce to hazardous OsO4 compound),22 and dissolved oxygen interference23,24 are major obstacles on working with the Os-complex

based

bio-electrocatalytic

[Mn2III(phen)4(O)Cl2]2+ complex in-situ

systems.

Herein,

we

report,

a

Mn-complex,

immobilized on carboxylic acid functionalized


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multiwalled carbon nanotube (f-MWCNT)/Nafion

system, designated as GCE/f-MWCNT-

Nf@Mn2III(phen)4O(Cl2), where phen=1,10 Phenanthroline, as an alternate, low cost, non-toxic, oxygen interference-free and high current sensitivity transducer for the GOx electrical wiring and efficient bio-electrocatalytic oxidation and sensing applications (316.7 A mM cm-2). Appropriate electrode surface modification of the transducer that can provide facile electrical communication with the enzyme, is a key step for the success of the bioanode development. In general, following methods have been adopted for the surface modification of the transducer: (i) Layer-by-layer formation; Quaternized heterocyclic PVP1-3,12,13,17 or PVI16,23 bearing Os(bpy)-complex is modified on anionic GOx layers; (ii) Grafting: Electro-reduction of amino-derivative of aryldiazonium salt on carbon surface followed by cross-linking with amino site of GOx using Poly(ethylene glycol) diglycidyl ether as a cross-linker (PEGDE; cross-linking between two amino-functional groups)24,25; (iii) Covalent immobilization: Amino-functionalized electro-organic system is coupled with carboxylic acid functionalized carbon nanotube via amide coupling;26 (iv) - interaction: Aromatic electro-organic compound, azo-dye (cango red) is physisorbed on sp2 carbon site (graphitic) of carbon nanomaterial modified electrode surface;27 (v) Self-assembled monolayer: Thiol functionalized electrochemical system is chemisorbed on gold electrode.14 (vi) In-situ polymerization: Pyrrole-momomer functionalized Os(bpy)-complex is electro-polymerized on solid electrode surface.11 Similarly, for the GOx enzyme connection with the redox mediator, ionic interaction between negatively charged GOx and positive charged Os(bpy)2-PVP/PVI,1-3,5,16,17 cross-coupling agent (PEGDE)15,20,28 and functional group activator (EDC/NHS; activate the carbonyl site for coupling of amino-functional group)14

have been

widely utilized. In this work, we introduce a simple and new strategy for highly efficient bioelectrocatalytic application of glucose, wherein, a transducer, [Mn2(phen)4(O)Cl2]2+ is in-situ



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prepared using Mn(phen)2Cl2 precursor on f-MWCNT followed by direct cross-linking with GOx enzyme without any linker and functional group activator. It is noteworthy that due to the natural abundance, appropriate redox chemistry and low toxicity, manganese metal and its complexes play a crucial role in biomaterials and electron transfer reactions.29,30 The 1,10phenanthroline ligand (phen) offers non-covalent immobilization strategies based on the π−π interactions between π-extended benzene rings and MWCNT sp2 carbon, and free Mn-Cl2 site for in-situ exchange with water molecule (Mn-OH2) and further for efficient electrical wiring with GOx enzyme moiety. The carboxylic acid functionalized MWCNT used in this work is not only providing π−π interaction, but also for strong hydrogen bonding and covalent coupling with amino-functional groups of the GOx.

2. EXPERIMENTAL 2.1. Reagents and materials. 1,10-phenanthroline, COOH functionalized multiwalled carbon nanotube (f-MWCNT; >8% COOH functional group, ~80% purity on carbon basis, size:9.5 nm×1.5μm) and 5% Nafion solution were purchased from Aldrich (USA) and diluted to 0.3% using ethanol and used throughout this work. MnCl2.4H2O was obtained from SD fine chemicals, India. Potassium iodate was purchased from Rankem, India. Other chemicals were of analytical quality grade and used as received. Screen-printed gold electrode (AuSPE) was obtained from Zensor R&D, Taiwan. A pH 7 phosphate buffer-supporting electrolyte was prepared by using distilled water of ionic strength 0.1 M phosphate buffer solution and used throughout this study.


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2.2 Instrumentation. Electrochemical analysis was studied with Metrohm Autolabs, Netherlands instrument. The three-electrode system consisting of chemically modified glassy carbon electrodes (GCE; Basi, Japan, 3 mm diameter, 0.0707 cm2 area) as a working electrode, Ag/AgCl as a reference electrode, and a platinum wire as an counter electrode. For transmission electron microscopy (TEM) analysis, FEI TECNAI G2 20-TWIN (Netherland) instrument was used. The discharge measurements of the biofuel cell are based on open-circuit voltage stability and polarization curve experiments. The loading voltage was set from high to low and low to high, by connecting the electrodes to an in-built range of external resistors. The polarization curve was obtained by calculating the current and voltage of the bio-fuel cell system. The highest point of the I-E curve indicated the high-power density of the fuel cell. A JASCO 4100 Spectrophotometer, Japan was used along with

KBr for FTIR analysis and Ocean Optics

instrument (JAZ-EL200-XR1) was used for the UV-Vis analysis. Similarly, a AZILTRON, PRO 532 (USA) instrument was used with a 532 nm laser excitation source for the Raman spectroscopic analysis. ESI-Mass analysis was carried with Q-Tof Mass Spectrometer Micromass instrument (Waters) and the Data processed using Mass Lynx NT software system. 2.3 Synthesis of Cis-[Mn(phen)2Cl2] Complex. The Mn-complex was synthesized by the reported methodology.31 Ethanolic solution of 1,10-phenanthroline (0.984 g. 6.3 mmol) in ethanol (30 mL), MnCl2.4H2O (0.5 g, 2.527 mmol) was mixed and allowed to stirred for 0.5 h. The obtained pale yellow coloured precipitate was filtered and washed with copies amount of methanol and vacuum dried. Calculated yield is 0.24 g (78 %). The electronic spectral data of the complex is 230 nm (120 M-1cm-1) and 347 nm (18275 M-1cm-1), which are coincide with the previously reported values.31 Measured m/z value using ESI-MS (in ethanol) is 450.09 [Mn(phen)2Cl]+ [M-Cl]+.



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Scheme 1. Cartoon of Mn(phen)2Cl2-Nafion complex (A); adsorption on a COOH functionalized MWCNT modified GCE (B); immobilization of the complex by - stacking and hydrogen bonding interaction (C) and its in-situ conversion to binuclear[Mn(phen)2O(Cl)2]2+ complex (D); its redox step elevation to high-valent complex (E); and the electrical wiring (F) in bio-electrocatalytic glucose oxidation reaction 2.4 Synthesis of GCE/f-MWCNT@Mn2(phen)4O(Cl)2-Nf@GOx electrode. In first, the GCE surface was cleaned mechanically and electrochemically by polishing with 0.5 micron alumina powder, washing with double distilled water and subjected to sonication for 5 min followed by cyclic voltammetry (CV) for 10 cycles in a potential window, -0.21 V vs


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Ag/AgCl

at

a

scan

rate

(v)

of

50

mV

s-1

in

pH

7

PBS.

Then,

GCE/f-

MWCNT@Mn2(phen)4O(Cl)2-Nf was prepared by a two-step (Scheme 1) method: In Step-I, about 5 μL aliquots of ethanolic suspensions/solutions of the f-MWCNT (1 mg/500 L), Mn(phen)2Cl2 (1 mg/500 L EtOH) and Nafion (0.3%) were drop casted on the GCE surface and dried at room temperature successively. Later, in step-II, an 5 μL aliquot of ethanol solution of the glucose oxidase (GOx) (3 mg/500 L) was drop casted and air dried (5±1 min). The resulting electrode was potential cycled in a window, −0.11.0 V vs Ag/AgCl for 10 cycles at v = 50 mV s−1 in pH 7 PBS. Such a procedure led to in-situ transformation of Mn(phen)2Cl2 precursor sample to [Mn2(phen)4(O)Cl2]2+ on the f-MWCNT adsorbed electrode surface (Scheme 1). A thin film of f-MWCNT@Mn(phen)Cl scratched out of from the above modified electrode was used for the FT-IR/KBr characterization. UV−Vis and ESI-MS characterizations were performed with an ethanol extract of as prepared GCE/f-MWCNT/[Mn2(phen)4(O)Cl2]2+ electrode used.

3. RESULTS AND DISCUSSION 3.1. In-situ immobilization of Mn(phen)2Cl2 complex. In first, bare GCE surface was used

for

the

electrochemical

studies.

Figure

1A

curve

a showed a typical CV response of Mn(phen)2Cl2-Nf solution drop-casted GCE, GCE/Mn(phen)Cl-Nf in pH 7 PBS. No faradic signal was noticed indicating the non-amenable feature of the Mn(phen)2Cl2 complex on the solid electrode. Interestingly, when, the Mncomplex-Nf solution is adsorbed on GCE/f-MWCNT, i.e., GCE/f-MWCNT@Mn(phen)Cl-Nf (modified electrode is tentative designated), a sharp redox peak at standard electrode potential, Eo’ = 590±20 mV vs Ag/AgCl with a peak-to-peak separation (Ep = Epa-Epc, where Epa and Epc



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B.

A.

C.

b. f-MWCNT@Mn(Phen)Cl-Nf A1

c. f-MWCNT@ Mn(Phen)Cl

100

50

50

0

0

C1

0 C1 v=50 mV s

0.4

0.8

0.0

-1

0.0

0.8

E/V vs Ag/AgCl

0

-100

ipc

Ep/V vs Ag/AgCl

100

0.9

Epa

0.8

Slope = 0.129

0.6

0

100

200

C.

1.2

60

pH 3-11

0 -30

Epc 1.2

300

v/mV s-1

0.8

30

Slope = 0.256

0.7

0.4

E/V vs Ag/AgCl F.

E.

D.

ip /A

-200

0.4

E/V vs Ag/AgCl

ipa

10-500 mV s-1

200

A1

a. Mn(Phen)Cl-Nf -50 on GCE

-50

0.0

f-MWCNT@Mn(Phen)Cl-Nf@GOx

I/A

I/A

100

1.6

2.0

2.4

log (v/mV s-1)

G.

2.8

-0.4

0.0

0.4

0.8

E/V vs Ag/AgCl

1.0

Epa/V vs Ag/AgCl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.8

0.6

Slope = -60 mV/pH

2

4

6

8

10

12

pH

Figure 1. (A) CV responses of Mn(phen)2Cl2-Nf system adsorbed on GCE (curve a) and GCE/f-MWCNT (curve b) and Mn(phen)2Cl2 adsorbed GCE/f-MWCNT (curve c) at v= 50 mVs-1 in PBS solution. (B) CV response of GOx enzyme immobilized on GCE/f-MWCNT@Mn2(phen)4(O)(Cl)2-Nf modified electrode. (C) Effect of scan rate and pH and (D and E) are plots of ipa and ipc vs scan rate and Epa and Epc vs log(scan rate) and (F) pH effect on CV response of GCE/f-MWCNT@Mn2(phen)4(O)(Cl)2-Nf@GOx at v=50 mV s-1. (G) Plot of Epa vs pH.; Inset scheme is a cartoon for the Mn-complex modified bio-electrode.


1.2

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are anodic and cathodic peak potentials respectively) value, 115±5 mV (Fig. 1A, curve b) were noticed. Calculated surface-excess (Mn) concentration is 5.46×10−9 mol cm−2. After the experiment, when the working electrode is gently washed with double distilled water and performed 20 continuous CV experiment in a blank pH 7 PBS, the redox peak was retained with a relative standard deviation (RSD) of 5% (data not enclosed). In addition, when the modified electrode was tested in N2 purged and normal dissolved oxygen containing pH 7 PBS, there is no difference in the redox response was noticed (data not enclosed) indicating the oxygen interference-free redox electrochemical response of the modified electrode. The electrode-toelectrode reproducibility was tested by preparing five modified electrodes at different timings showing a RSD = 8.9% (data not enclosed). The slightly higher RSD value obtained over normal value 5% is due to the manual preparation error.

Meanwhile, as a control experiment,

Mn(phen)2Cl2 dissolved ethanol solution (without Nafion) was modified on GCE/f-MWCNT, i.e., GCE/f-MWCNT@Mn(phen)Cl and was tested for redox feature (Figure 1A. Curve c). Qualitatively same voltammetric response, but, with poor stability (RSD = 26%) was noticed. This observation indicated that the anionic exchanging Nafion serves as a protective layer for the metal complex without altering its redox feature. In

next,

GOx

enzyme

modified

GCE/f-MWCNT@Mn(phen)Cl-Nf,

GCE/f-

MWCNT@Mn(phen)Cl-Nf@GOx was prepared by adsorbing a dilution solution of GOx on the Mn-complex modified electrode. Figure 1B is a twenty cycles CV response of the GCE/fMWCNT@Mn(phen)Cl-Nf@GOx in pH 7 PBS. A stable voltammetric response (RSD=5%) was noticed. Compared with the GCE/f-MWCNT@Mn(phen)Cl-Nf, the GOx modified electrode showed about 30% reduction in the redox peak current signal (Figure 1A, curve b and Figure 1B), which may be due to the interaction effect of the insulating protein-layer of the GOx with



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the underlying surface. In order to evaluate the electro-kinetic parameters, the bio-electrode was further subjected to scan rate studies as in Figure 1C. A systematic increase in the peak current with linear increase in the scan rate was observed. Calculated peak currents (ipa and ipc) and peak potentials (Epa and Epc) were plotted against v as in Figure 1D and E. A linear line starting from origin was noticed (ip vs v plot) illustrates adsorption controlled electron-transfer phenomenon of the bio-anode. Plots of Epa and Epc vs logv yielded slope values 129 and 135 mV dec-1 respectively. Applying Laviron theory and its equation,32 the charge transfer coefficient, α and apparent electron transfer rate constant, ks can be calculated: SLa/SLc = α /1-α

(1)

log ks = α log (1-α) + (1-α) log α - log [RT/nFv] – α (1-α)nFΔEp / 2.3RT

(2)

Where, SLa = slope of the linear plot of Epa vs logv (0.135), SLc = slope of the linear plot of Epc vs logv (0.129 V) and α = charge-transfer coefficient and other variables have its own implications. Calculated α and ks are 0.69 and 1.56 respectively. Although the obtained ks value is comparable with standard redox systems, 1.34 (hematin)33 and 1.3 (heme),34 the obtained  value is slightly higher than the ideal case, the 0.5 values implies the non-ideal surface and electron-transfer feature of the bioanode. The redox reaction is found to be proton-coupled electron-transfer in nature and obeys Nernstian type E-pH relation (-60 mV pH-1) (Figure 1F and G). At this stage, it is difficult to conclude what kind of Mn-complex is immobilized and what is the structure of the f-MWCNT@Mn(phen)Cl-Nf@GOx. In addition, since, the amount of Mncomplex active site immobilized on the modified electrode is very small (~ng), it is a challenging task to characterize the system. A systematic effort has been taken to find out the true-active site


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of the modified electrode using several physicochemical characterization techniques and several control experiments.

B. f-MWCNT@Mn(phen)Cl f-MWCNT@Mn(Phen)2(H2O)2

A.A.f-MWCNT f-MWCNT A. f-MWCNT

B.

C.

D.

a. f-MWCNT

b. f-MWCNT@Mn(phen)Cl

b. Mn(Phen)2Cl2

ID

315

ID/IG = 0.32

c.f-MWCNT@Mn(phen)Cl

1425

290

%T a.f- MWCNT

1620

276

244

IG

Intensity/AU

ID/IG = 0.54

Intensity/AU

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b.f-MWCNT@Mn(phen)Cl

350 a. Mn(phen)2Cl2

478

1200

1500 -1

cm

1800

3000

2000

1000

-1

cm

200

400

600

/nm

Figure 2. Comparative (A) TEM images of f-MWCNT and f-MWCNT@Mn(phen)Cl. (B) Raman spectra of fMWCNT (curve a) and f-MWCNT@Mn(phen)Cl (curve b). (C) FTIR spectra data of f-MWCNT (curve a), Mn(phen)2Cl2 (curve b) and f-MWCNT@ Mn(phen)Cl (curve c). (D) UV-Vis spectra of Mn(phen)2Cl2 (curve a) and f-MWCNT@ Mn(phen)Cl (curve b) modified bio-electrode. For the UV-Vis, an ethanolic extract was used for the analysis.



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3.2. Physicochemical characterization of f-MWCNT@Mn(Phen)Cl. Figure 2A shows the comparative TEM images of f-MWCNT and f-MWCNT@Mn(phen)Cl illustrates the agglomeration of the molecular system on the basal plane of the f-MWCNT. It is likely that the - interaction between the aromatic electrons of phen-ligand and sp2 carbon of the f-MWCNT turn the composite as an agglomerated system. Figure 2B is a comparative Raman spectroscopy response of f-MWCNT and f-MWCNT@Mn(phen)Cl modified screen-printed gold electrodes (to avoid confusion in the characterization, Nafion layer is omitted) showing an decrease in the intensity ratio of disordered (D) and ordered graphitic (G) bands from 0.54 to 0.32 (I = ID/IG) after the immobilization of the Mn-complex. Plausible reason for the observation is due to the  assisted immobilization of the phen ligand of the Mn-complex on f-MWCNT and its enhanced graphitic feature of the composite. Figure 2C is a comparative FTIR responses of f-MWCNT, Mn(phen)2Cl2 and f-MWCNT@Mn(phen)Cl. Specific IR signals at 1620 cm-1 and 1425 cm-1 vibrations analogous to v(C=C) and v(C=N) stretching of 1,10-phenanthroline ligand with in the Mn(phen)2Cl2 (curve b) was noticed.35 When the complex is modified by in-situ electrochemical method on f-MWCNT, part of the FTIR signals were retained but with marked shift in the frequency (curve c) indicating the specific interaction between the Mn-complex and underlying f-MWCNT surface.36 In addition, feeble IR response at 550-800 cm-1 was also noticed speculating -oxo type functional group formation and some structural changes in the Mncomplex on the f-MWCNT surface.37 Figure 2D, curve b shows the UV-Vis spectra of an extract of GCE/f-MWCNT@Mn(phen)Cl in ethanol solution showed distinctly different pattern against the precursor Mn(phen)2Cl2 complex (Fig. 2D, curve a). The precursor Mn(phen)2Cl2 complex displayed characteristic peaks at 244, 290 and 350 analogous to the π−π* transition of the aromatic phen ligand (244, 290) and metal-to-ligand charge transistion (MLCT) of the


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Manganese(II)bis(1,10-phenanthroline) chloride complex.38 Whereas, the electronic spectra of ethanolic solution of GCE/f-MWCNT@Mn(phen)Cl (Fig. 2D, curve a) exhibits a peaks at 239, 276 and 315 nm along with a low energy broad absorption peak at 478 nm due to transitions between molecular orbitals composed of orbital dπ of Mn and pπ of the oxo-bridge existing of a typical of oxo-ligand of a Mn(phen) dimers, MnIII-µ-Ooxo-MnIII.39-41 In order to confirm the true Mn-complex species, ESI-Mass spectroscopy analysis of ethanloic solution of the modified electrode was analysed in comparison with the precursor complex [MnII(phen)2Cl]+ (ca. mol. wt. = 450.09) as in Supporting Information Figure S1A and B. A specific m/z peak at 459.04, which is corresponding to the [Mn2III(Phen)4(O)(Cl)2]2+ (ca. mol. wt. = [M+2H]+ = [M]/2 = 459.04))42 and a major m/z peak at 451.14 which is due to the fragmentation of the above complex as [MnII(phen)2(H2O)2]2+ complex (ca. mol. wt. = 451.14) were noticed confirming the existence of MnIII-µ-Ooxo-MnIII type of complex on the f-MWCNT@Mn(Phen)2Cl system. In further, based on the standard electrode potentials (Eo) of the Mn redox states,43 plausible redox reaction noticed with the f-MWCNT@Mn(Phen)2Cl are depicted as a MnIII-O-MnIII/MnIV-OMnIV

redox state (Eo’=0.489 V vs Ag/AgCl).44 Overall, it is proposed that under electrified

condition at GCE/f-MWCNT surface, the Mn(phen)2Cl2 complex gets converted to [Mn2III(Phen)4(O)(Cl)2]2+ like intermediate, which has been further trapped by anionic (sulfonic acid of Nafion), - (graphitic structure), hydrogen bonding sites (carboxylic functional group of f-MWCNT) of the electrode (Scheme 1). This point onward the modified electrode used in this study re-designated as GCE/f-MWCNT@Mn2(Phen)4(O)(Cl2)-Nf, wherein, Mn2(Phen)4(O)(Cl2) = [Mn2(Phen)4(O)(Cl2)]2+.



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e. d. b.

Slope = 0.111 V

0.4 -6

-5 log(I/A)

100 50

with 2 mM Glu

7

1/2

0.5

c.

-1

a.

ipa/v /A (V s )

0.6

ipa/A

E/V vs Ag/AgCl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

0 0.0

0.2 1/2

0.4

6 0

-1 1/2

100

200

v/mV s

v / (V s )

300

-1

C.

B.

A. C. b + 2mM Glu

+0-6 mM Glu f-MWCNT@Mn2(phen)4O(Cl)2-Nf

b.f-MWCNT@Mn2(phen)4O(Cl)2-Nf@GOx

A1 c.f-MWCNT@Mn2(phen)4O(Cl)2-Nf+Glu

v= 2mVs 0.0

-1

5 A

C1

0.4 E/V vs Ag/AgCl

0.8

100 A

0.0

0.8 E/V vs Ag/AgCl

1.6

v=10 mVs 0.0

20 A

-1

0.4

0.8

E/V vs Ag/AgCl

Figure 3. (A) CV responses of GCE/f-MWCNT@Mn2(phen)4(O)(Cl)2-Nf@GOx without (curve a) and with 500 µM of Glucose (curve b) in pH 7 PBS at solution v = 2 mV s−1. Inset (a) is a Tafel plot. (B) Effect of CV scan rate on the bio-electrocatalytic oxidation of 2 mM glucose on GCE/f-MWCNT@Mn2(phen)4(O)(Cl)2-Nf@GOx. Inset plots are; (b) ipa vs v1/2 and (c) ipa/v1/2 vs v. (C) CV responses of GCE/f-MWCNT@Mn2(phen)4(O)(Cl)2-Nf@GOx with various concentrations of glucose from 0 to 6 mM at v = 10 mVs−1. Inset schemes (d) and (e) are cartoon for the GCE/fMWCNT@Mn2(phen)4(O)(Cl)2-Nf@GOx and its electron-transfer reaction respectively.

3.3. Bioelectrocatalytic oxidation and sensing of glucose. Figure 3A curves a and b are CV responses of the GCE/f-MWCNT@Mn2(Phen)4(O)(Cl2)-Nf@GOx without and with 500 M of glucose at scan rate=2 mV s-1. A well-defined oxidation signal starting at 0.4 V with a peak maximum, i.e., peak at anodic peak potential, Epa = 0.55 V vs Ag/AgCl, where the A1/C1 redox peak exist. As a control experiment, f-MWCNT@Mn2(Phen)4(O)(Cl2)-Nf (without GOx) was


Page 17 of 30

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tested with 500M of glucose, but, failed to show any faradic response (curve c) showed the bioelectrocatalytic activity of the GOx modified electrode. In order to confirm the electron-shuttling between the GOx and Mn-complex active site, discreet electrocatalytic glucose oxidation experiment was carried out under N2 and O2 gas purged pH 7 PBS as in Figure 4. Note that GOx enzyme can oxidize glucose in couple with dissolved oxygen with formation of H2O2 as an intermediate species (1st generation glucose biosensing).9 If there is an influence of dissolved oxygen on the electrocatalytic oxidation, then, with the N2 gas purged reaction condition, a marked decrement in the glucose electrocatalytic response than that of the dissolved O2 reaction condition will be noticed. On the other hand, if

b&c. a+5 mM glu-N2 atm/O2 atm 40

20

I/A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0

-20

a. f-MWCNT@Mn(bpy)OCl-Nf@GOx N2 atm. 0.0

0.2

0.4

0.6

0.8

1.0

E/V vs Ag/AgCl

Figure 4. Typical CV responses of GCE/f-MWCNT@Mn2(phen)4(O)(Cl)2-Nf@GOx for 2 mM glucose oxidation reaction at N2 and O2 purged pH 7 PBS at v= 10 mV s-1. Note: Mn2(phen)4(O)(Cl)2= [Mn2(phen)4(O)(Cl)2]2+.

any absence of current variation without and with O2 purged will be observed, it supports the electron-shuttling reaction. Figure 4 is the typical CV response of the bioanode response for the



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electrocatalytic oxidation of glucose at N2 and O2 purged conditions showing absence of any remarkable changes in the peak current signal. This observation confirms the molecular wiring and the electron-transfer shuttling reaction without any oxygen-interference. The increasing part of a steady-state (I-E) curve obtained in Figure 3A, curve b is further subjected to Tafel slope analysis as in inset Figure 3(a). A Tafel slope value (bc = ∂E/∂logI; 2.303RT/nF) value, 111±1 mV decade-1 was obtained. By assuming number electron-involved in the rate-determining step is 1, calculated  = 0.53. This value is closer to an ideal value (0.5) indicating symmetry energy barrier for the electron-transfer reaction. Effect of scan rate showed a linear increase in the peak current starting form origin indicating the diffusion-controlled reaction mechanism for the glucose oxidation reaction (Figure 3(b)). Figure 3(c), is a plot of scan rate normalized current value (ipa/v1/2) against the v showing a parabolic curve, wherein, higher current value at low v and nominal value at high v were observed indicating the coupled chemical reaction mechanism with involvement of electron-transfer step followed by chemical reaction as shown in the inset Schemes in Figure 3(d) and (e). In fact, the result verifies the electron-transfer shuttling reaction of the Mn-complex between the GOx enzyme and underlying electrode surface. Figure 3C is CV response of the bioanode with increasing concentration of glucose (0-6 mM) at v=10 mV s-1. A plot of anodic peak current (ipaGlu; base-line corrected) vs [glucose] is linear in a concentration window 06 mM with regression coefficient (R) and current sensitivity of 0.9997 and 24.7 µA mM-1 (349.4 µA mM-1 cm-2) respectively (data not shown). In further stability of the bioanode was tested by fifty continuously CV cycling with 2 mM of glucose at a v=50 mV s-1 (Supporting information Figure S2). Slight decrement in the first 3 cycles after that nearly stable voltammetric response was noticed. The bio-electrocatalytic performance remains same for up to 3 days (data not shown) indicating good stability of the working electrode. In further, other Mn-


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complex, Mn(bpy)Cl2 modified electrode, prepared similar to the Figure 1A, was subjected to CV and bio-electrocatalytic oxidation of glucose in pH 7 PBS as in (Supporting information Figure S3). Although the redox response is very similar to the Mn-Phen complex base system, with respect to the electrocatalytic activity and calibration graph, the Mn-bpy complex modified bio-electrode showed poor response. Although precise reason for the observation is unclear, it is expected that three aromatic units in the Phen ligand provides better - interaction with underlying graphitic carbon than that of the two aromatic units containing bipyridyl ligand and in turn for the better redox and bio-electrocatalytic activities. In next, amperometric i-t technique was adopted for the bio-electrocatalytic sensing of the glucose at a fixed applied potential, Eapp = 0.65 V vs Ag/AgCl (optimal) and at hydrodynamic condition as in Figure 5A curve a. Successive spikes of 50 µM of glucose resulted to regular increase in the current values. The amperometric i-t signals are linear against the glucose concentration in a window, 50550 M with a current sensitive value = 22.4 µA mM-1 (or 316.8 µA mM-1 cm-2) (Figure 5B). For the first ten spikes of glucose showed a RSD = 4.1%. Calculated detection limit value (S/N=3) = 6.7 M. It is important to highlight that the current sensitivity value noticed in this work is 280,000 times higher than that of the values noticed with several Os-complex based bioanodes; poly(benzoxazine) modified with Osmium complexes (0.004 A mM cm-2)18, Os(bpy)-PVI assembled by layer-by-layer on SWCNT (3.4A mM cm2),13

aldehyde and carbonyl groups bearing Os-complexes (12.5 A mM cm-2),21 gold

nanoparticle/Sodium dodecylsulfate

based Os-complex system (28 A mM cm-2),20 3-



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0.8

A.

B.

i/A

50M

50M

0

10

20 -1

[Glucose] /mM

8

30

-2

15

-1

10

5 Slope = 22.4 A mM

b.

0

C.

Cell voltage / V

5

0.0

a.

10

Slope: 0.033 Intercep: 0.028

Eapp = 0.65 V vs Ag/AgCl

15

0.4

400

800

0 0.0

t/s

0.2

0.4

6

0.2

4

0.1 2

-1

0.6

0.8

0.0

Power Density / W cm

-1

i /A

-1

LB Plot:

I/A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

0

0

[Glucose]/mM

20

40

60

80

100 -2

Cell Current Density/A cm

Figure 5. (A) Comparative amperometric i-t responses of GCE/f-MWCNT@Mn2(phen)4(O)(Cl)2-Nf@GOx (curve a) and GCE/f-MWCNT-Nf@GOx (curve b) for successive detection of 50 µM of glucose at an applied potential of +0.65 V vs Ag/AgCl in pH 7 PBS. (B) Typical calibration response of glucose bio-electrocatalytic current vs glucose concentration. Inset is a plot of i-1 vs [Glu]-1. (C) Fuel cell power output and cell voltage in PBS 7 containing 2 mM glucose and saturated dissolved O2. Inset cartoon is an illustration of the bio-fuel cell system.

ferrocenylpropyl-modified linear polyethylenimine (LPEI), 6-ferrocenylhexyl-modified LPEI and poly[(vinylpyridine)Os(bipyridyl)2Cl]2+/3+ (~70 A mM cm-2)15 and Osmium BipyridineContaining Redox Polymers Based on Cellulose (176.8 A mM cm-2). 17 The amperometric i-t data is further extended to calculation of Michaelis-Menten (MM) kinetics for the glucose oxidation reaction using the following Lineweaver-Burk (LB) plot: 45

1/i =KM/nFAkc ΓMn [Glu]+1/nFAkcΓMn =SLLB [Glu] INLB SLLB=KM/nFAkc ΓMn;

INLB= 1/nFAkc ΓHematin

(3) (4)

Wherein, i= glucose oxidation current signal, [Glu]=Concentration of glucose; ΓMn = total surface concentration of the electro-active Mn-complex site (4.1×10-9 mol cm-2); KM =


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Michaelis-Menten rate constant; kc = catalytic rate constant (first-order) for the glucose oxidation reaction; SLLB = Slope of the LB plot; INLB = intercept of the LB plot and other symbols have its usual significance. Inset Figure 5 is a LB plot showing SLLB and INLB values 0.033 M A-1 and 0.0281A-1 respectively. The MM kinetics parameters were calculated as follows; KM = 1.18 mM dm-3; kc = 1.27 s-1. The heterogeneous electron-transfer rate constant value, k’ME is then obtained by substituting the other parameters, kc, Mn and KM in k’ME = kc×Hb/KM as 4.4×10-3 cm s-1. Obtained KM value (1.18 mM) is comparable with the value, 2.32±0.1 mM on various Os(bpy)-PVP/PVI based bioanode systems.46 In addition, the GCE/f-MWCNT@Mn2(Phen)4O(Cl)2-Nf@GOx bioanode is extended to enzyme biofuel cell application in couple with oxygen reduction reaction at Pt (cathode site) in oxygenated pH 7 PBS (Figure 5C and inset Scheme). The bio-electrochemical cell was immersed in 2 mM glucose dissolved pH 7 PBS and the cell voltage is monitored using the potentiostat. Consequently, the biofuel cell was successfully polarized by connecting the electrodes to a range of variable external resistors. Figure 5C shows the plot of cell voltage and the power density against the current density. As can been seen, a peak power density value 7.3 W cm−2 at opencircuit potential, Eopen =0.25±0.1 V vs Ag/AgCl was noticed. This result is comparable and/or superior than that of the recently reported miniature enzymatic biofuel cells based on various Os(bpy)-transducers like; Os(dmo-bpy)2 pendant-poly(4-vinylpyridine) modified Indium-TinOxide (ITO) (0.7 W cm-2; Eopen= 0.38 V vs Ag/AgCl ),47 dodecylsulfate

gold nanoparticle/Sodium

based Os-complex system (3.5W cm-2; Eopen= 0.45 V vs Ag/AgCl)20 and

osmium-bis-(2,2′-bipyridyl)-PVI modified graphite electrode (1.5W cm-2; Eopen= ~0.4V vs



Page 22 of 30

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Ag/AgCl).48 Although, this new system showed a high current sensitivity, observation of about 200 mV positive redox potential over the Os(bpy)-based complex is a limitation of this system.

4. CONCLUSION A highly redox active and stable electrochemical immobilization of Mn(phen)2Cl2 complex on carboxylic acid functionalized glassy carbon modified electrode was achieved. The modified electrode showed a distinct electron-transfer reaction with standard potential, Eo’ = 0.59 V vs Ag/AgCl in pH 7 PBS. The redox system was found to be adsorption controlled electron-transfer feature with pH dependent property. The immobilized Mn-complex on the modified electrode surface is revealed by performing various physicochemical characterizations using TEM, Raman, IR and UV-Vis spectroscopy and ESI-MS as [Mn2III(phen)4(O)(Cl)2]2+ complex. The GOx enzyme modified electrode, GCE/f-MWCNT@Mn2(phen)4O(Cl)2-Nf@GOx was

prepared

by

simple

adsorption

of

a

dilute

solution

of

GOx

on

GCE/f-

MWCNT@Mn2(phen)4O(Cl)2-Nf. The bioanode showed well-defined bio-electrocatalytic glucose oxidation current with a Tafel slope of 111 mV dec-1. Amperometric i-t based electrochemical sensing of glucose showed a current linearity in the concentration window, 50550 M with current sensitivity value, 316.7 A mM cm-2 , which is about 280,000 times higher than the Os-complex modified bioanode systems, indicating improved electrical wiring of the Mn-complex with GOx enzyme active site. Michaelis-Menten kinetics equation was further applied and estimated the kinetics parameters as KM = 1.18 mM dm-3; kc = 1.27 s-1; k’ME = 4.4×10-3 cm s-1. Using this new system, biofuel cell operation, wherein, the GOx modified electrode for glucose oxidation reaction bioanode coupled Pt cathode for oxygen reaction was constructed and achieved a power density value 7.5 W cm-2.


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AUTHOR INFORMATION Corresponding Author Annamalai Senthil Kumar*, Emails; [email protected]; [email protected]; Phone: +91-416-2202754 ORCID Annamalai Senthil Kumar: 0000-0001-8800-4038 Notes The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was funded by Department of Science and Technology, Science Engineering Research Board, India.

ASSOCIATED CONTENT Supporting Information ESI-Mass

of

response

of

ethanolic

solution

of

Mn(Phen)2Cl2

and

f-

MWCNT@Mn2(phen)4(O)(Cl)2 (Figure S1), stability of the working electrode (Figure S2) and CV responses of GCE/f-MWCNT@Mn(bpy)2(H2O)2-Nf@GOx with various concentrations of glucose (Figure S3). The Supporting Information is available free of charge on the ACS publications website.



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ACKNOWLEDGMENT The authors acknowledge the DST–SERB, Scheme (DST-SERB-EMR/2016/002818) for the financial support of this work. NS acknowledges the DST-SERB, national postdoctoral fellowship (PDF/2015/000253) program.

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