Unprecedented Wiring Efficiency of Sulfonated Graphitic Carbon

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Unprecedented wiring efficiency of sulfonated carbon nitride materials: towards high-performance amperometric recombinant CotA laccases biosensors Alain R Puente-Santiago, Daily Rodríguez-Padrón, Xuebo Quan, Mario J. Muñoz Batista, Ligia O. Martins, Sanny Verma, Rajender S. Varma, Jian Zhou, and Rafael Luque ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b05107 • Publication Date (Web): 23 Nov 2018 Downloaded from http://pubs.acs.org on November 23, 2018

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ACS Sustainable Chemistry & Engineering

Unprecedented wiring efficiency of sulfonated carbon nitride materials: towards high-performance amperometric recombinant CotA laccases biosensors Alain R. Puente-Santiago,[a]* Daily Rodríguez-Padrón,[a]+ Xuebo Quan,[b]+, Mario J. Muñoz Batista,[a] Lígia O. Martins,[c] Sanny Verma,[d] Rajender S. Varma,[e], Jian Zhou,[b]* Rafael Luque[a, f]* aDepartamento

de Química Orgánica, Grupo FQM-383, Universidad de Cordoba, Campus de

Rabanales, Edificio Marie Curie (C-3), Ctra Nnal IV-A, Km 396, E14014, Cordoba (Spain), e-mail: [email protected], [email protected] b

School of Chemistry and Chemical Engineering, Guangdong Provincial Key Lab for Green

Chemical Product Technology, South China University of Technology, Guangzhou 510640, P. R. China, e-mail: [email protected] c

Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa Av

da Republica, 2780-157, Oeiras, Portugal. dOak

Ridge Institute for Science and Education, P. O. Box 117, Oak Ridge TN, 37831, USA

eRegional

Centre of Advanced Technologies and Materials, Department of Physical Chemistry,

Faculty of Science, Palacky University, Šlechtitelů 27, 783 71 Olomouc. fPeoples

Friendship University of Russia (RUDN University), 6 Miklukho-Maklaya str., 117198,

Moscow, Russia

ACS Paragon Plus Environment

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+These

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authors contributed equally to the work.

Abstract Controlling electron transfer (ET) processes across electrochemically active biomaterials by tuning the surface properties of platform materials plays a key role towards the design of highly efficient biosensors. In this work, ET rates of recombinant CotA laccases have been drastically improved by an immobilization process on sulfonic groups modified graphitic carbon nitride (Sg-CN) materials. Cyclic voltammetry (CV) and fourier transform IR (FTIR) spectroscopy revealed that the enzymes undergo striking conformational changes onto graphitic carbon nitride (g-CN), adopting an electrochemically inactive configuration, while retain their native-like structure with a superb ET efficiency on Sg-CN surfaces. In fact, the resulting CotA laccase/SgCN biomaterial displayed an electron transfer (ET) rate constant of (12 ± 0.5) s-1, the highest value reported to date for a direct electron transfer reaction of multicopper oxidases attached to carbon-based materials. Importantly, the combined parallel tempering Monte Carlo (PTMC) and all-atom molecular dynamics (AAMD) theoretical calculations proved CotA incorporation in a highly ordered array with an overall positive surface density composed of lysine and arginine domains in contact with net negative charged Sg-CN surfaces, which promoted a 1200 fold improvement of the free enzyme ET rate constant. An ET pathway has been putted forward taking into account the orientation of CotA laccase on the Sg-CN surface. Additionally, CotA laccase/Sg-CN biomaterial was tested as an amperometric biosensor delivering outstanding bioelectrocatalytic activities toward the oxidation of catechol and syringol, relevant emerging pollutants. In fact, the sensitivities of the CotA laccase/Sg-CN/ITO electrodes were 0.95 A M−1 cm−2 and 0.41 A M−1 cm−2 for catechol and syringol, respectively, surpassing mostly of the laccase biosensors reported in the literature.


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INTRODUCTION Electron transfer (ET) processes from enzymes redox groups to electrodes have been widely investigated owing to their significant impact for the design and development of enzyme-based sensors,1,

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bioelectronic systems3,

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or biocatalytic cells.5,

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Specially, the design of hybrid

electrically active biomaterials towards the fabrication of highly efficient electrochemical biosensors with improved electronic properties, is still a current challenge. In this regard, several strategies have been attempted to construct novel biosensors that exhibit efficient electronic wiring between redox proteins and electrodes.7 Among them, the incorporation of carbon based materials that promote a good electronic wiring of redox proteins and the use of genetically engineered enzymes, which facilitate the most efficient ET pathways, have been successfully employed.8-11 Jonathan Claussen and coworkers have developed an electrochemical biosensor, which utilize single-walled carbon nanotubes (SWCNTs) modified with Au-coated Pd nanocubes to enhance the electrocatalytic activity for glucose detection.12 Qion Zen et al. have constructed a hydrogen peroxide amperometric biosensor with enhanced performances through the electrostatic assembly of sodium dodecyl benzene sulphonate (SDBS) functionalized graphene sheets (GSs) and horseradish peroxidase (HRP).13 Gorton and coworkers have intensively investigated the effect of the cysteine mutations on both the ET behavior and bioelectrocatalytic activity of HRP immobilized on gold surfaces.14,

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Noticeably, electrodes functionalized with

recombinant HRP exhibited a high sensitivity for H2O2 of 2.0±0.1 A M-1cm-2, which is several times larger than most of the hydrogen peroxide electrochemical biosensors reported in the literature.16,

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Despite high-performance biosensors can be reached by applying the former


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approaches, up to now, a highly sensitive amperometric biosensor combining the two strategies has not been reported. Among the multicopper oxidases, laccases constitute redox enzymes which are able to control the one-electron oxidation of four substrate equivalents with the electro-reduction of dioxygen to water.18 All the members of the family contain at least a mononuclear blue type 1 copper centre (T1), close to the surface, which is involved in substrate oxidation, and a trinuclear centre (T2/T3) at 12-13 Å distant, in which the reduction of dioxygen to water is produced. Among them, CotA laccase is a principal constituent of the outer coat layer of Bacillus Subtilis and is well-known by its outstanding thermostability properties. It should be pointed out that the crystal structure of CotA laccase was first elucidated by Lígia O. Martins and coworkers.19 In this work, a novel hybrid electrically active biomaterial with a remarkable ET efficiency was achieved through the electrostatic assembly of recombinant bacterial CotA laccases on sulfonated graphitic carbon nitride surfaces. Such biomaterial was deposited on indium-tin oxide (ITO) electrodes to construct a high-performance amperometric biosensor. The obtained electrode exhibited highly desirable properties in a linear range of 1-900 µM for trace amounts determination of pollutants such as phenol derivatives, which have been recognized as dangerous waste materials from industries.20 In fact, the aforementioned pollutants have been ranked by the United States Environmental Protection Agency (EPA) the 11th in the list of 126 toxic chemicals and consequently has been designated as priority pollutants.21 The role of the sulfonic group layer on the electrochemical properties of the immobilized CotA laccase was unravelled through the electrochemical and spectroscopic characterization obtained from the enzymes anchorage onto modified and unmodified carbon nitride surfaces, respectively, as well as by all-atom molecular dynamic simulations of the CotA laccase/Sg-CN biomaterial. Unlike most of the


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works published, in this contribution, we connect using FTIR, XPS, electrochemistry and molecular dynamics techniques, the structural properties of multicopper oxidases immobilised onto carbon-based materials (conformational and orientation states) with both their electron transfer (ET) and bioelectrocatalytic function. EXPERIMENTAL SECTION Materials Graphitic carbon nitride and sulfonated graphitic carbon nitride materials were synthesized following procedures previously reported.19,

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Catechol, syringol and other chemical were

purchased from Sigma-Aldrich. All stock solutions were obtained using deionized water from a Millipore water purification system. Production of the recombinant CotA laccase The enzyme CotA-laccase from Bacillus subtilis was heterologously produced in Escherichia coli Tuner (DE3) pLacI strain (Novagen) using the pLOM10 plasmid (Martins et al. 2002). The recombinant cells growth and enzyme purification were performed as previous described. 24, 25

Preparation of Cot A laccase/g-CN and CotA laccase/Sg-CN biocomposites g-CN and Sg-CN (4 mg) were diluted in deionized water (2 mL). The obtained dispersions (2 mL, 2 mg mL− 1) were mixed with CotA laccase solution (2 mL, 4 mg mL−1) at pH=5.5. The resulting composite was sonicated (15 min) and then maintained at 4 °C overnight with shaking. Afterwards, the mixtures were centrifuged at 12000 rpm for 10 min to obtain a white solid. Deionized water (2 mL) was added and the resulting biomaterial was redispersed by a moderate shaking. The aforementioned protocol was repeated three times to guarantee the total removal of


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the content of free or physically adsorbed enzymes. The obtained biocomposites were then diluted in 1 mL of deionized water and stored at 4 °C in a cold room. Preparation of the electrodes ITO electrodes were sequentially cleaned and sonicated with deionized water, ethanol and acetone respectively. Subsequently, the dispersions (30µL) of Cot A laccase/g-CN and CotA laccase/Sg-CN were drop casted onto ITO surfaces and dried at room temperature. Characterization of the biomaterials CotA laccase/g-CN and CotA laccase/Sg-CN were characterized by Fourier Transform-Infrared Spectroscopy (FT-IR) and X-ray Photoelectronic Spectroscopy (XPS) to attain a full structural characterization of the enzymes as well as their elemental composition in the obtained biomaterials. FTIR analysis was accomplished on the ABB MB3000 infrared spectrophotometer with a window of ZnSe, an ATR PIKE MIRacleTM sampler and 256 scans at a resolution of 16 cm-1. Spectra were recorder employing the Horizon MBTM software in a 4000-400 cm-1 wavenumber range, at room temperature. The amide I bands were fitted following previous works.26 XPS measurements were carried out using an ultrahigh vacuum (UHV) multipurpose surface analysis system SpecsTM with the Phoibos 150-MCD energy detector. The experiments were accomplished at pressures

Unprecedented Wiring Efficiency of Sulfonated Graphitic Carbon

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