Electrodeposition of nanoMnO2 from mineral leach liquor and the investigation on conformational changes of hemoglobin induced by the nanomaterial

Journal Pre-proof Electrodeposition of nanoMnO2 from mineral leach liquor and the investigation on conformational changes of hemoglobin induced by the nanomaterial Ayonbala Baral, Lakkoji Satish, Subrat Kumar Padhy, Dipti P. Das, Bankim Chandra Tripathy, Malay K. Ghosh

PII:

S0927-7757(19)31094-5

DOI:

https://doi.org/10.1016/j.colsurfa.2019.124102

Reference:

COLSUA 124102

To appear in:

Colloids and Surfaces A: Physicochemical and Engineering Aspects

Received Date:

5 August 2019

Revised Date:

8 October 2019

Accepted Date:

10 October 2019

Please cite this article as: Baral A, Satish L, Padhy SK, Das DP, Tripathy BC, Ghosh MK, Electrodeposition of nanoMnO2 from mineral leach liquor and the investigation on conformational changes of hemoglobin induced by the nanomaterial, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2019), doi: https://doi.org/10.1016/j.colsurfa.2019.124102

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Electrodeposition of nanoMnO2 from mineral leach liquor and the investigation on conformational changes of hemoglobin induced by the nanomaterial Ayonbala Barala,b, Lakkoji Satishd,e, Subrat Kumar Padhy,a,b Dipti P Dasa,c, Bankim Chandra Tripathyb, Malay K. Ghosha,b* a

Academy of Scientific and Innovative Research (AcSIR), CSIR- IMMT Campus, Bhubaneswar, Odisha - 751013, India b

Hydro & Electrometallurgy Department, CSIR- Institute of Minerals and Materials Technology Bhubaneswar - 751013, Odisha, India c

Central Characterization Department, CSIR- Institute of Minerals and Materials Technology, Bhubaneswar, Odisha - 751013, India Department of Chemistry, National Institute of Technology Rourkela, Odisha -769008, India

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Department of Chemistry, Ravenshaw University, Cuttack, Odisha - 753003, India

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*Corresponding author

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Dr. Malay K. Ghosh, Hydro & Electrometallurgy Department, CSIR- Institute of Minerals and Materials Technology, Email – [email protected], Phone - +91 6742379374

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Graphical abstract

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We also investigated the thermal unfolding and refolding of HB in presence of NPs has been investigated using CD spectroscopy which depicts that the ellipticity values of HB in the buffer medium remain intact.

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UV-visible spectra of HB show heme peak in presence of both NP which illustrates that there was no significant spectral change of the Soret band in intensity, position, and shape. This indicates that in presence of NPs, HB structure is not disturbed.

Abstract The electrodeposition of manganese dioxide from purified leach liquor obtained by reduction leaching of polymetallic manganese nodules using sucrose as a reductant in H2SO4 medium followed by 2 stages purification process was described. The role of cationic additive on the morphology and electrochemical properties were investigated by using Cetyltrimethylammonium bromide (CTAB) (0–500 mg dm−3). The orthorhombic phase of γ-MnO2 was confirmed from the XRD patterns of as prepared electrolytic

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manganese dioxide (EMD). The effect of CTAB on EMD has been investigated in terms of morphology, current efficiency (CE) and energy consumption. In addition to this, for the first time we have studied the interactions of EMD with hemoglobin (Hb). Herein, the molecular interactions between Hb and nano EMD prepared at an optimum condition were explored using various spectroscopic techniques including UV-vis absorption, fluorescence, and circular dichroism. Various quantitative parameters

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such as association/dissociation constants, binding cooperativity were determined to illustrate the biomolecular interactions. Our results suggest that hemoglobin remains structurally stable in the

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bioconjugates formed by the simple adsorption method. However, at higher temperature, hemoglobin was found to adopt an unfolded conformational state both in free form and bioconjugates. This work

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would be helpful in designing of safe nanoparticles (NPs) for biomedical applications.

Introduction

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Keywords: nano-MnO2; electrodeposition; biomolecular interactions, nanomaterial; leaching

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Manganese (Mn) is the 10th most abundant element in the Earth’s crust and can easily oxidise near earth’s surface forming ˃ 30 known Mn oxidehydroxide minerals. The mineralogy and geochemistry

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of Mn in the upper crust strongly depend on these oxides.[1] Approximately 90–95% of the current Mn production is consumed in the metallurgy of iron and steel industries as an alloying element.[2] Currently, the use of manganese as electrolytic manganese dioxide (EMD) and chemical manganese dioxide (CMD) in the primary and secondary battery industries has been significantly increased.[3] The growing demand for manganese has made it so important to develop processes for the economic recovery of Mn from manganese bearing natural resources.[4] Seabed nodule has been considered as a potential source for valuable metals like manganese (Mn), copper (Cu), cobalt (Co), and nickel (Ni). This is also called as polymetallic nodule or manganese nodule, spreading over 30% of the ocean floor

forming nodular concentric circles by entrapping varieties of metals in the framework.[5, 6] Several processes have been developed across the globe for the extraction of these base metals economically.[7, 8] Manganese oxides have gained much attentions in a wide range of applications such as hybrid electric vehicles, fuel cells, super capacitors, water remediation, biomedicine and pharmaceutical industries owing to specific physicochemical properties.[9, 10] Among these, 1D-nano manganese dioxide (MnO2) such as nanotubes, nanorods, nano fibers, and nanowires have been exponentially used for several applications[11] due to their unique characteristics like large surface area, good stability, and mainly, the possibility of application in batteries and more recently in the field of biomedicine, drug delivery and photocatalytic reactions.[12-14] Synthesis of nano MnO2 with controlled morphology,

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crystallographic form, shape and architecture has been considered as the key research area to establish well defined properties such as; optical, catalytic, magnetic and mechanical. MnO2 exhibits several polymorphs like α-, β-, γ-, δ-, and ԑ-types based on the arrangements of octahedrons [MnO6] by edgesharing. [15-17], EMDs are basically γ- type and predominately used in battery industries.[18]

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Biswal et.al developed a process for the extraction of Mn as EMD from Mn sludge obtained through ammonia–ammonium sulfate– sulfur dioxide leaching of Mn nodules and studied the discharge

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capacities of the prepared EMD samples.[4] In one of our previous works, the extraction of Mn from polymetallic nodule through reductive leaching using sucrose as a reducing agent in the sulphuric acid medium was described.[5] The leach liquor obtained through the above process is treated as the source

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for MnO2 deposition. Two stages of purification process for the generation of purified MnSO4 solution from nodule leach liquor was reported somewhere else.[19] The discharge properties of EMD both in the presence and absence of cationic surfactant (CTAB) were studied which revealed that the discharge

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capacity of MnO2 was enhanced in presence of CTAB. In accordance with one of the publications of Ghaemi et.al, the increase in electrochemical property was related to the adsorption of surfactants at the electrode/electrolyte interface. They explained that the mass transport properties of materials increase

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after surface modification which directly influenced crystalline structure, size, mechanical properties,

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and activity of materials.[20]

With the emergence of nanoscience in biomedical research and applications, it is very much essential to estimate the consequences of biomolecular interactions for improved bioavailability of NPs, biodistribution, and safety. There are several reports available in the literature on the molecular interactions between NPs and proteins.[21, 22]. However, very few of those studies are based on nanoMnO2. Due to the nontoxic nature, MnO2 NPs find applications in different biological studies.[23, 24] Moreover, these NPs have been applied in biosensing field. In one study, He. D et al. reported a new biosensing platform for probing and recognizing biomolecular interactions by attaching DNA to nanoMnO2.[25] Also, few MnO2 nanosheet assisted biosensor have been developed based on

florescence properties. [26, 27] In spite of the applications in biological field, there are no systematic studies to understand the molecular interactions between proteins and nanoMnO2 which will be helpful in the design of biocompatible nanomaterials. In our previous studies we reported the effect of nano MnO2 and MnO2@RGO nanocomposites on the structure and stability of bovine serum albumin.[28, 29].

In this study, to assess the biological importance of EMD, we investigated the molecular interactions of EMD with hemoglobin (HB) for the first time. HB is an essential element in the vascular system of animals which also acts as an oxygen carrier, directly and indirectly, transports carbon dioxide and regulates the blood pH.[30] The basic function of HB is to remove hydrogen ions in the capillaries and

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to carry them to the lungs. Moreover, it is involved in many clinical diseases such as leukemia, anemia, heart disease, excessive loss of blood, etc.[31] The molecular weight of HB is 64,500 kDa and it contains four globin chains, among which two are α- chains, and two are β- chains.[32] HB molecule has four oxygen-binding sites along with α- chains containing 141 amino acids and β chains containing 146 amino acids.[33] Fluorescence quenching has been extensively used for the interaction studies of

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biomolecules, but so far the molecular interactions between EMD and hemoglobin have not been reported as per our knowledge. The effect of EMD on the conformation of HB has also been analyzed

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by means of circular dichroism and UV-vis spectroscopy. Immobilized proteins are found to be stable than the free proteins in solution. However, at high temperature, proteins loose structure stability and activity. Therefore, refolding of thermally denatured proteins is desirable. Refolding of small proteins

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on solid supports are reported in earlier studies.[34] In this context, we investigated the thermal unfolding and refolding of HB in presence of EMD. HB is very interesting for refolding studies because the recovery needs each subunit to be properly folded and assembled into a quaternary structure with

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the incorporation of the prosthetic group at its native site.

Figure 1. Crystal structure of HB (PDB-2HHB)

Experimental Procedures Materials

HB (Product No- A2153) was purchased from Sigma-Aldrich and used without any further purification. Indian Ocean nodule (Polymetallic metallic nodule) used for this work was collected from National Institute of Oceanography (NIO) Goa, India. The chemical composition of the nodule was obtained through wet chemical analysis after crushing and grinding of nodules. The metal contents are; Cu 0.93%, Ni 1.17%, Co 0.09%, 67 Mn 24.4%, Fe 6.5%. The other chemicals used in this study were of analytical grade and purchased from Thermo Fischer Scientific, India. Double distilled water was used for the preparation of phosphate buffer (pH 7.4). Methods

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Electrodeposition of MnO2 from purified Solution The preparation of EMD was discussed in detail somewhere else from purified leach liquor obtained through reductive leaching of Mn nodules in presence of sulphuric acid and sucrose as a reductant.[19] The dissolution process of Mn from polymetallic nodule was reported in one of our previous studies.[5] All the electrodeposition experiments were carried out for 6h in a 500 mL glass beaker containing

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purified leach liquor as electrolyte, pure lead (Pb) as anode and stainless steel (SS) 304 grade as cathode, placed in a thermostatic water bath to maintain the electrolytic bath temperature. The constant DC

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source was used by regulated power supplier [0–30 V, 10A, DC Power supply, APLAB, INDIA]. The anodic current efficiency (CE) was calculated from the weight gained by anode at the end of electrolysis. A multimeter was assigned to measure the cell voltage and current at regular intervals. The effect of

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CTAB on CE and EC were examined by varying the CTAB concentration in the electrolytic bath during deposition of MnO2. The deposited MnO2 was washed several times with ethanol, water and dilute acid

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for the removal of surface active reagent and dried in an oven at 80 0C overnight.

Sample Preparation for biomolecular interaction studies A buffer solution, consisting of 0.01 M sodium phosphate at pH 7.4, was used in all the experiments.

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The Protein-NP solutions were prepared by adding a fixed amount of nanoparticle solution to protein solution. The solutions were vortexed followed by incubation at room temperature for 2 hours. Two

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different nanoparticles have been used such as blank EMD (NP1) and EMD in presence of CTAB (NP2).

Characterization of EMD The as-obtained EMD samples were characterized by different analytical techniques. The phase identification of the electrodeposited samples were carried out using X-ray diffraction (XRD) (Model: PAN ANALYTICAL PW 1830). The surface morphology of the deposited nano MnO2 was investigated

by field emission scanning electron microscopy (FESEM) (Model: Zeiss, Supra55 model), transmission electron microscopy (TEM) (model: FEI, TECNAI G2 20, TWIN) operated at 200 kV. Characterization of biomolecular interactions between EMD and HB Fluorescence Spectroscopy The fluorescence measurements were carried out using Horiba Jvon Spectrometer (Fluoromax-4P) with excitation wavelength at 295 nm to avoid the interference from Tyr (tyrosine) and Phe (Phenylalanine). The fluorescence spectra were collected as a function of wavelength in the range of 305 to 450 nm. Excitation and emission slit widths were fixed at 5nm for each. Fluorescence intensities were corrected by subtracting the appropriate blank samples (without protein). The protein concentration was fixed at

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0.4 mg/ml and NP concentration was varied from 0 – 0.08 mg/ml. The fluorescence intensities are corrected for absorption of the exciting light and/or reabsorption of the emitted light by the molecules present in the solution to eliminate the inner filter effect using following equation [35] Icor = Iobs e(Aex +Aem)/2

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Where, Icor and Iobs are the corrected and observed fluorescence intensities, respectively, and Aex and

Circular Dichroism (CD) Spectroscopy

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Aem are the absorption values of NP at the excitation and emission wavelengths, respectively.

The CD spectra were obtained using a JASCO spectropolarimeter equipped with a thermostat-

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controlled cell holder. The nanoparticle concentration was varied from 0 – 0.2 mg/ml by keeping protein concentration constant at 0.2 mg/ml. The spectra were taken in a wavelength range of 200-260 nm with an average of three scans and a bandwidth of 1 nm. The final spectra were obtained by subtracting the

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spectra of background (without protein) from the original protein spectra. Spectra were deconvoluted to estimate secondary structure content with the DICHROWEB service using the SP175 dataset and the CDSSTR analysis program.[36-38] For thermal unfolding and refolding studies, each sample was

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subjected to a heating cycle of 25-90-25 0C at a rate of 10 0C/min.

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UV-Vis absorption measurements UV-Vis absorption spectra of HB in presence of NPs were recorded in Agilent UV-Vis spectrophotometer (Model: Cary 100) using 1 cm path length quartz cuvettes. The concentration of NP was varied from 0 – 0.08 mg/ml. Results and discussion Leaching and Purification

The dissolution of metals was performed and optimized in our previous work through leaching of polymetallic nodules under mild conditions. The optimized condition for getting a maximum percentage of metal recovery was found to be: 10% pulp density (PD), 10% (v/v) H2SO4, 7% (w/w) sucrose at 90 °C. The maximum metal extraction was achieved in 2h and the percentage of metal recovery: Mn, > 99%; Ni, 98%; Cu, 87%; Co, 83%. The overall reaction of manganese dissolution or reduction in presence of sucrose as a reductant in the sulfuric acid medium was expressed as;[5] 24𝑀𝑛𝑂2 + 𝐶12 𝐻22 𝑂11 + 24𝐻2 𝑆𝑂4 = 24Mn𝑆𝑂4 + 12CO2 + 35𝐻2 O

The leach liquor obtained from nodule leaching was subjected to adsorption by activated charcoal for

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30min to remove the organic content in the solution. The two-stage purification process was established for the removal of impurities like; Co, Ni and Cu from the nodule leach liquor.[19] The 1st stage purification process involved removal of iron as Fe(OH)3 by Ca(OH)2 addition to increase the solution pH from 2.5 to 4.0. For the 2nd stage purification process, sodium sulfide(Na2S) was added (3 to 4 times more than that of the stoichiometry requirement for Cu, Ni, Co, and Zn) to separate Co2+, Ni2+, Cu2+,

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Zn2+ as their respective insoluble sulfides and the final pH of the solution was ~ 6.

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Preparation of EMD

The percentage ratios of Mn and H2SO4 was maintained as 2:1 by adding sulfuric acid to the purified

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solution before electrodeposition of manganese dioxide. The deposition of manganese dioxide is basically, an anodic oxidation process of MnSO4 solution at an inert electrode surface. The overall cell reaction of the electrolysis process can be represented as[3, 39]

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Anode: 𝑀𝑛2+ + 2𝐻2 𝑂 ⟶ 𝑀𝑛𝑂2 + 4𝐻 + + 2𝑒 − (𝐸 0 = −1.224𝑉𝑣𝑠 𝑆𝐻𝐸) Cathode: 2𝐻 + + 2𝑒 − ⟶ 𝐻2 (𝐸 0 = 0𝑉 𝑣𝑠 𝑆𝐻𝐸)

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Overall: 𝑀𝑛2+ + 2𝐻2 𝑂 ⟶ 𝑀𝑛𝑂2 + 2𝐻 + + 𝐻2 (𝐸 0 = −1.224𝑉𝑣𝑠 𝑆𝐻𝐸) The process parameters viz. current density, temperature, concentration of sulfuric acid was optimized

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prior to study the effect of CTAB in the said solution during electrodeposition of manganese dioxide. From column-1 (Table-1), it is observed that with an increase in current density from 100 to 200 A.m−2, at temperature 90°C using 50% sulfuric acid of the concentration of manganese(II) ion, the current efficiency increased and reached to a maximum 68.03% along with decrease in energy consumption to 2.459 KWh.kg−1. However, further increase in the current density decreased the current efficiency and increased energy consumption. Column-2 (Table-1) shows that by reducing the temperature from 90°C to 80°C, extra amount of manganese(II) ion get oxidized at the anode and hence, the current efficiency increased from 68.03 to 71.19% resulting in decrease in energy consumption from 2.459 to 2.236

KWh.kg−1. With further decrease in temperature, a decreasing trend in current efficiency was observed. The concentration of sulfuric acid was varied within a range of 30% to 60% to the concentration of Mn(II) ion in the raffinate and shown in column-3 (Table-1). It was found that with change in sulfuric acid percentage from 30% to 50% the CE increased and EC decreased but on further increase in H2SO4 concentration, the CE decreased along with slightly increase in EC. The current density was varied again with optimized temperature (80°C) and concentration of sulfuric acid (50%) shown in column-4 of table 1. This depicts that the CE increased and EC decreased with increase in current density from 100 to 200 A.m−2 but beyond 200 A.m−2, a decreasing trend of CE and increase in EC was observed. However, the CE and EC were found to be 71.19% and 2.236 KWh.kg−1 respectively at an optimized current density of 200 A.m−2. Table 1, summarized that the optimum conditions for electrodeposition

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of manganese dioxide is; CD: 200 A.m−2, Temp: 80°C, H2SO4: 50% of [Mn2+]. Table 1. Effect of temperature, current density, conc. of sulfuric acid during electrodeposition of manganese dioxide from sulfate solutions Temp: 90°C, H2SO4: 50% of [Mn2+]

CD: 200 A.m−2 H2SO4: 50% of [Mn2+]

CD: 200 A.m−2 Temp: 80°C

*CD (A.m−2)

CE (a)

EC (b)

*Temp (°C)

CE (a)

EC (b)

*[H2SO4] (#%)

CE (a)

100

62.41

2.61

90

68.03

2.459

30

53.33

200

68.03

2.459

80

71.19

2.236

40

300

65.65

2.698

70

69.45

2.151

400

63.14

2.943

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Temp: 80°C, H2SO4: 50% of [Mn2+] *CD (A.m−2)

CE (a)

EC (b)

2.766

100

67.41

2.083

60.97

2.5

200

71.19

2.236

50

71.19

2.236

300

68.52

2.426

60

65.21

2.318

400

64.33

2.823

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EC (b)

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*Variable parameter, #: % of manganese concentration in the electrolyte; a: %, b: KWh.kg −1

Further investigations were carried out to study the effect of CTAB on the electrodeposition of manganese dioxide from sulfate solution at an optimized condition. The concentration of CTAB was

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varied from 0 to 500 mg. dm−3to evaluate the effectiveness of the additive and the results are shown in figure 2. However, it has been previously reported that in presence of organic additives the CE increases

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and the EC decreases.[19] The presence of additive in the electrolytic bath helps to maintain the surface tension over the growing electrode surface and facilitates smooth and compact deposition by organizing and regulating the position of adatoms on the site of growing surface. The adhesion of depositing substance is very strong with the substrate in presence of additive which is reverse in case of deposits obtained in absence of additive.[20] CTAB is widely used as organic additives in many electrodeposition processes.[40, 41] In our case, a maximum CE of 71.19 % was achieved in absence of additive while in presence of 10 mg.dm-3CTAB, the current efficiency increased to 75.55% and the energy consumption decreased from 2.236 to 2.001 KWhkg−1. In the presence of CTAB concentration

at 100 mg. dm−3, CE increased to 87.64% with a further decrease in EC to 1.703 KWhkg-1. This may be due to increase in surface adsorption of the additive on the electrode surface which ultimately leads to a uniform surface deposit and increased CE. However, with further increase in concentration of CTAB, there was a significant decrease in current efficiency. In presence of 500 mg. dm−3 CTAB, the resulted CE is found to be 70.64% and EC 2.322 KWhkg-1.

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CE (%)

80 2

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1.8

70

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100 200 300 400 500 −1 Concentration of additive (mg.L )

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EC

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CE

EC (kWh.Kg−1 )

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Figure 2. Effect of CTAB on CE and EC during electrodeposition of EMD from manganese nodule solution. CD: 200 A/m2, [H2SO4]: 50% of the concentration of manganese (II) ion, temperature: 80°C

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XRD

XRD patterns of electrolytic manganese dioxide both in the absence and presence of CTAB are shown in Figure 3. The XRD patterns of the electrodeposited manganese dioxide corresponds to an

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orthorhombic γ phase of MnO2 with lattice constants, a = 6.366 Å, b = 10.15 Å, and c = 4.089 Å, indexed to the Joint Committee on Powder Diffraction Standards (JCPDS) card No. 14-0644. [15] In

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all the case the order of crystal orientation remain as (120), (131), (300), (160), (421). The XRD patterns of the deposited MnO2 remain unaltered irrespective of the concentration of CTAB (0 to 500 mg. dm−3) in the electrolytic bath during deposition. The purity and crystallinity of the assynthesized products are confirmed from the crystal planes.

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Intensity (a.u)

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III

20

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421

160 50

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2 (degree)

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300

120

131

II

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Figure 3. XRD pattern of MnO2 electrodeposited in presence and absence of CTAB. CD: 200 A/m2, [H2SO4]: 50% of the concentration of manganese (II) ion, temperature: 80°C. I: Blank, II: 10 mg. dm−3, III: 50 mg. dm−3, IV: 100 mg. dm−3, V: 250 mg. dm−3, VI: 500 mg. dm−3

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FESEM

FESEM images of electrodeposited MnO2 in the presence and absence of CTAB are shown in Figure 4. Figure 4(a and b) represents the surface morphology of blank EMD where, it is observed that the

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growth of particles are not well organized and form agglomeration. The deposit contains both unsymmetric spherical layered aggregates along with rod-like structures develope from the aggregate with no distinct grain boundaries. The coarse and irregular deposits may be obtained due to the

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formation of MnO2 crystal nucleus during reaction progress. Ghaemi et.al reported that in sulfate medium, MnSO4 reacts with H2SO4 first and form MnO2 nuclei as the reaction continuance in the absence of surfactant. In the presence 50 mg dm−3 CTAB, the urchin like appearance was observed from the lower magnification image (Figure 4c). The higher magnification image describes that the more prominent rods are united to form urchins (Figure 4d). Figure 4e depicts that in presence of 100 mg dm−3 CTAB, MnO2 forms well organized regular fiber like growth with distinct grain boundaries. The high magnification image (Figure 4f) of EMD at 100 mg dm−3 CTAB reveals that the presence of CTAB directed the nano-particles to grow in a dwarf hairgrass type manner (moss carpet structure), where all

the single units are rooted from the same flat base.[19] In presence of 100 mg dm−3 CTAB, the surface of the deposit is of highly arranged, less agglomerated with well distinct grain boundaries (Figure 4e and 4f). On further increase in concentration of CTAB (250 mg dm−3) does not have significant change in the growth patterns of the grains (Figure 4g and 4h) but the size of the particles are smaller as compared to the size of MnO2 obtained in presence of 100 mg dm−3 CTAB. Further increase in the concentration of CTAB beyond 250 mg dm−3 upshots, highly porous deposits with less adhesive grains of unspecified grain boundaries (figure 4i and 4j). From the above studies, we conclude that the presence of CTAB, irrespective of the concentrations in the electrolytic bath is able to control the formation of agglomeration. CTAB helps to grow the grains in a particular direction with an evenly distribution manner on the electrode surfaces which results, smooth deposit on the surface of the elctrode. The X-

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ray mapping of EMD (Figure 4k and 4l), shows the uniform distribution of both Mn and oxygen (O) on the deposited surface. The EDX results (figure 4m and 4n) represents that MnO2 formed in this process is highly pure.

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1 µm

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200 nm

f

1 µm

200 nm

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200 nm

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200 nm

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1 µm

200 nm

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1 µm

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Figure 4. FESEM images of manganese dioxide electrodeposited in presence and absence of CTAB; a: Blank (1µm), b: Blank (200nm) c: 50 mg.dm−3 (1µm), d: 50 mg.dm−3 (200nm), e: 100 mg.dm−3 (1µm), f: 100 mg.dm−3 (200nm), g: 250 mg.dm−3 (1µm), h: 250 mg.dm−3 (200nm), i: 500 mg.dm−3 (1µm), j: 500 mg.dm−3 (200nm), k: l: m: n: TEM

The morphology and structure of the prepared MnO2 was investigated using transmission electron microscopy (TEM) and high resolution TEM (HRTEM). The TEM micrographs (Figure 5) indicates that the size of the particles are in nano range. In absence of CTAB the development of nano rods are less and nonuniform, ranges from (20-35) nm in length and (9-12) nm in width (Figure 5a). The presence of 100mg.dm-3CTAB, directed the nanoparticles to grow in an uniform manner forming the fibrous morphologies of size (20-25) nm in length and (˂ 10) nm in width (figure 5e). The HRTEM images of as-synthesized γ-MnO2 demonstrates that the nanoparticles are polycrystalline in nature (Figure 5b-5c and 5f-5g). Irrespective of the CTAB concentration, the crystal planes of nanoparticles remain same which indicates additive has no role on the crystal structure. The SAED patterns of bare MnO 2 and MnO2 in presence of 100mg.dm-3 shown in figure 5d and 5h which confirms that the plane of orientation

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of nano particles are in (131), (300) and (160) directions which are also in good agreement with the XRD results. The HRTEM images of fringes obtained for EMD in absence of CTAB are shown in figure 5b and 5c and for EMD in presence of 100mg.dm-3 CTAB are shown in figure 5f and 5g respectively. It indicates that unrelatedly the concentration of CTAB, the interplanar distance between the fringes is 0.24 nm, referred the growth of crystal plane along [131] direction which is in accordance

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with XRD observations.

Figure 5. TEM image of bare γ-MnO2 (a), HRTEM image of bare γ-MnO2 (b & c), SAED pattern of γ-MnO2 (d), TEM image of γ-MnO2 in presence of 100mgdm-3 CTAB (e), HRTEM image of γ-MnO2 in presence of 100mgdm-3 CTAB (f & g), (h) SAED pattern of γ-MnO2 in presence of 100mgdm-3 CTAB Molecular interactions between EMD and hemoglobin

Fluorescence quenching efficiency The intrinsic fluorescence properties are extremely sensitive to the microenvironment of fluorophores. The fluorescence emission spectra of HB were monitored with excitation at 295 nm, which selectively excites the tryptophan residues. We investigated the binding affinity, binding sites, dynamics and conformational changes of HB from the fluorescence study. It is observed from figure 6 that HB shows emission maxima at 328 nm and the fluorescence intensity gradually decreases with increasing NP concentration. The quenching by NPs suggests the direct interaction between NP and the chromophore residues of the HB. It could also be due to the HB adsorption onto NP which causes shortening of the distance between the quencher and the fluorophores. In addition, there was no shifting in the emission

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microenvironment of tryptophan residues upon adsorption.

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maximum wavelength (λmax) in presence of NPs at all concentrations, which indicates the unaltered

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Figure 6. Representative fluorescence emission spectra of HB in the absence and presence of NPs with increasing concentrations (0.002, 0.004, 0.008, 0.02, 0.04, 0.08 mg/ml). a) NP1 and b) NP2

The efficiency of fluorescence quenching of HB by the NPs was derived based on the Stern–Volmer

𝐼0

= 1 + 𝐾𝑆𝑉 [𝑄]

(1)

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𝐼

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model, as explained in equation (1),[42]

Where, I0 and I represent the maximum emission intensity of HB in the absence and presence of NP, respectively, KSV is the Stern–Volmer quenching constant signifying the fluorescence quenching efficiency and [Q] is the concentration of the NP. Based on the mathematical fitting of the experimental data to equation (1), the Stern–Volmer quenching constants were calculated (Figure 7). Interestingly, we noted that the quenching efficiency of NP1 was higher than NP2.

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Figure 7. Fluorescence quenching efficiency of HB by NPs. Stern–Volmer plots of the fluorescence quenching of HB by NP1 (a) and NP2 (b) Binding of HB to NPs

The relative strength and cooperativity of the NP–protein interactions were examined by quantifying

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several key parameters which can describe the association between HB and NP, i.e., binding dissociation constant (KD), binding association constant (KA), and the Hill coefficient (n). Using the

re

non-linear curve fitting of the fluorescence quenching of HB by the NPs, the key parameters were determined based on the Hill equation, as represented in equations (2) and (3),[43]

𝑄 𝑄𝑚𝑎𝑥

(𝐼 0 −𝐼)

(2)

𝐼0

=

lP

𝑄=

[𝑁𝑃]𝑛

(3)

𝑛 +[𝑁𝑃]𝑛 𝐾𝐷

na

where Qmax is the saturation value of Q, KD is the equilibrium binding dissociation constant which describes the relative strength of the NP–protein interaction, and n represents the Hill coefficient which

Jo

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defines the cooperativity of the NP–protein association.

Figure 8. Fluorescence quenching properties of HB by NCs and the associated NC–protein complexes binding parameters. Hill plots of the fluorescence quenching of HB in the presence of NC1 (a) and NC2 (b) with increasing concentrations. Experimental data were fitted based on the Hill mathematical model as represented by eqn (2) and (3). The estimated equilibrium dissociation constant, KD, of the NP–HB protein complexes are shown in table 2. It is observed that the dissociation constant of NP2-HB complex was comparatively higher than NP1-HB complex. From the KD value, we quantified the equilibrium binding association constant, KA. As expected a strong association between NP1 and HB is noticed. The experimental results on the NPHB binding interactions agreed with the fluorescence quenching efficiency of the HB by NP. In fact, the HB-NP binding interaction data are useful to explain the stronger fluorescence quenching efficiency

the NP (higher KA value). Table 2. Summary of the NP–HB protein binding parameters

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of NP1. The higher KSV value could be due to the closer proximity of the fluorophores to the surface of

NP2

KSV (mL mg−1)

69.76±4.27

35.71±3.26

Qmax

1.0568±0.0852

n

1.5039±0.3075 0.0105±0.0020

−1

98.6893±17.4546

KD (mg mL )

1.8407±0.1329 0.0146±0.0008 67.4400±4.3525

lP

KA (mL mg )

1.0069±0.0283

re

−1

-p

NP1

To understand the cooperative nature of binding between the NPs and HB, Hill constants, n for the NPHB complexes were determined from the fluorescence quenching data based on eqn (2) and (3). It

na

describes the binding cooperativity or the degree of independence of molecular interactions between multiple binding sites. From table 2, it is clear that cooperative binding (n > 1) is observed for both the complexes. This suggests that a single protein molecule adsorption onto the NP surface leads to higher

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association of further protein molecules to the NPs. The positive cooperativity of the NP-HB interactions could be attributed to the multiple binding sites of HB protein on NP.

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Conformational stability

In most cases adsorption of protein onto the surface of nanoparticles leads to structural changes or denaturation of tertiary and/or secondary structures of protein. However, some proteins retain their native structure after adsorption. It is well known that the surface chemistry of the nanomaterials influences the conformational stability of proteins which subsequently makes proteins biologically inactive.[21, 44] Various studies displayed the drastic conformational changes of proteins after adsorption onto the NP surfaces. Out of several NPs, MnO2 NPs are very less explored and observed to

have a positive effect on proteins. In our pervious study, we found that the molecular interactions of

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bovine serum albumin with MnO 2 NP produced insignificant structural changes.[42]

Figure 9. CD spectra of HB in presence of NP1 (a) and NP2 (b) with concentrations (0, 0.002, 0.02, 0.2 mg/ml) at 25 °C. Free NP solutions without protein are represented as NP1 and NP2 with concentrations 0.002, 0.02, 0.2 mg/ml. In this study, we utilized CD spectroscopy to know the effect of NPs with different concentrations on

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the secondary structures of the hemoglobin. In the far-UV region, HB exhibited two negative bands centered at 208 nm and 222 nm (Figure S1 provided in supplementary information). The secondary

re

structural content of HB was calculated using DICHORWEB services and provided in table 3. It is observed that the helicity of HB remains intact in presence of both the NPs except at high concentration

lP

(0.2 mg/ml), where 15 % loss in case of NP1 and 10% loss in case of NP2 are noticed. Table 3. Estimated secondary structure content as determined form CD spectra using DICHORWEB service Beta

Turns

Unordered

NRMSD

0.64

0.09

0.08

0.2

0.008

0.002

0.63

0.07

0.08

0.22

0.011

0.02

0.64

0.08

0.1

0.17

0.005

0.2

0.54

0.11

0.1

0.26

0.012

0.002

0.65

0.07

0.1

0.18

0.008

0.02

0.64

0.09

0.11

0.14

0.005

0.2

0.58

0.1

0.12

0.2

0.008

(NP)

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HB+NP1

ur

HB

HB+NP2

na

Concentration Alpha

Moreover, to assess the impact of the NPs on the 3-dimensional global conformation of the HB, UV/Vis spectroscopy was employed. The absorption spectra of HB exhibit several peaks at 279 (aromatic residues), 349 (ε band), 406 (heme/Soret band), 540, and 576 nm (oxy-band) (Figure 10).[45] HB in

native state produces a strong Soret band (due to π-π* electronic transition) at 406 nm. It is ascribed to the heme group embedded in a hydrophobic pocket.[46] Any changes in the shape and position of the Soret band provide valuable information on protein’s conformational changes. UV-visible spectra of HB show heme peak in presence of both the NPs. The spectra of HB in presence of NPs were background corrected by subtracting the respective absorbance of the NPs at each concentration. The absorption spectra of NP solutions in absence of HB are given in the supplementary information (Figure S1). It is noteworthy that there was no significant spectral change of the Soret band in intensity, position, and shape. This indicates that in presence of NPs, HB structure is not disturbed drastically and retains its stable native form where the heme group is not exposed to the aqueous medium.

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We also investigated the thermal unfolding and refolding of HB in presence of NPs to illustrate the effect of NPs on refolding of unfolded HB using CD spectroscopy. With the increase in the temperature to 90 0C, HB begins to denature due to the breakage of intra-protein hydrogen bonds which maintain its native state. The spectra of HB in presence of NPs during thermal unfolding and refolding are provided in supplementary information (Figure S2). Herein, we studied the unfolding and refolding of HB by

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taking ellipticity values at 222nm. Figure 11 shows the ellipticity value at 222 nm of HB in the absence

Jo

ur

na

lP

re

and presence of NPs during thermal unfolding and refolding measurements.

Figure 10. UV-visible spectra of HB showing heme peak in presence of NP1 (a) and NP2 (b) with concentrations (0, 0.002, 0.004, 0.008, 0.02, 0.04, 0.08 mg/ml) at 25 °C.

It is evident that the ellipticity values at 222 nm of HB in the buffer medium and also in the presence of the NPs, remain intact, which suggests the native structure of HB. In the buffer medium, the ellipticity value decreased drastically when the temperature was raised. On cooling to room temperature, it was observed that HB was unable to refold back to the native state. Also, in presence of NPs refolding was hardly observed. Only in presence of higher concentrations (0.2 mg/ml), a slight increase in the

lP

re

-p

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ellipticity values observed, which indicates HB is able to refold back slightly upon adsorption to NPs.

Conclusion

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Figure 11. Ellipticity value at 222 nm of HB in the absence and presence of NPs during thermal unfolding and refolding studies. a) NP1 and b) NP2. [Blue- Initial 250C, Orange- 900C, Grey- Final 250C]

The production of EMD was attained through 3 stages such as; leaching, purification, and

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electrodeposition. The morphological data reveal that surface of the blank EMD is dull, irregular and coarse. The presence of 100mg.dm-3 CTAB in the elctrolytte favored the nanoparticles to grow in a disciplined manner which results in uniform and bright deposits. Presence of 100mg.dm-3 CTAB

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enhances the current efficiency and lowers the consumption of energy. Further increase in CTAB concentration in the electrolytic bath, adversely affects the CE and EC. The optimum conditions derived for the electrodeposition of EMD from purified nodule solution in absence of CTAB is; Temp: 80°C, H2SO4: 50% of [Mn2+], CD: 200A.m−2 at which the CE and EC are 71.19% and 2.236 KWhKg-1 respectively. At optimum parameters in presence of 100mg.dm-3 CTAB, the CE increased to 87.64% with a further decrease in EC to 1.703 KWhkg-1. Based on the electrochemical properties and morphological studies, we can conclude that the electrodeposition of MnO2 in presence of 100 mg.dm3

CTAB was best amongst all.

The protein structural changes in the HB-EMD NP conjugates were characterized by fluorescence, CD, and UV-vis spectroscopic techniques. Our results demonstrate that the secondary and tertiary structure of HB in the bioconjugates remains intact. CD results suggested a slight loss of helical structure at higher concentration of NP. Declaration of Conflict of Interest

On behalf of all the co-authors of this manuscript I, Malay K. Ghosh as a corresponding author as an authorized person take the responsibility in declaring that there is no conflict of interest in submitting the manuscript as whole or the data contained in it for publication purpose.

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Malay K. Ghosht Acknowledgment

Authors are thankful to Prof. Suddhasatwa Basu, Director, CSIR-Institute of Minerals and Materials

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Technology for his kind permission to publish this work. One of the authors (A. Baral) is thankful to Ministry of Earth Sciences, Govt. of India, New Delhi for providing Senior Research Fellowship and the raw material during the course of the investigation. Authors acknowledge Dr. Harekrushna Sahoo

re

for his support in terms of the facilities to carryout circular dichroism experiments at NIT Rourkela, India.

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