An antimicrobial enzymatic biofuel cellXinxin Xiao*,a, Michael P. Ryanb, Dónal Leechc, Jingdong Zhanga, Edmond Magner*,b

aDepartment of Chemistry, Technical University of Denmark, Kongens Lyngby 2800,

DenmarkbDepartment of Chemical Sciences and Bernal Institute, University of Limerick, Limerick,

IrelandcSchool of Chemistry & Ryan Institute, National University of Ireland Galway,

University Road, Galway, Ireland

Corresponding authors: Xinxin Xiao, E-mail: xixiao@kemi.dtu.dk; Edmond Magner, E-

mail: edmond.magner@ul.ie

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Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2020

Experimental section

Materials

D-(+)-glucose (99.5%), 3,4-ethylenedioxythiophene (EDOT, 97%), GOx from

Aspergillus niger (EC 1.1.3.4, type II, ≥15 U mg-1), Myrothecium verrucaria BOx (EC

1.3.3.5, 15-65 U mg-1), Luria Bertani (LB) broth (containing 5 g L-1 NaCl, 10 g L-1

tryptone and 5 g L-1 yeast extract), sodium phosphate (monobasic dehydrate ≥99% and

dibasic ≥99%), ampicillin sodium salt (Amp) and sulfuric acid (H2SO4, 95-98%) were

obtained from Sigma-Aldrich, Ltd. Os(bpy)2PVI was synthesised via an established

procedure [1, 2]. All solutions were prepared using deionised water (18.2 MΩ cm, Elga

Purelab Ultra, UK).

NPG films[3, 4] were fabricated by etching 100 nm thick Au/Ag leaf alloy leaves (12-

carat, Eytzinger, Germany) in concentrated HNO3 (Sigma-Aldrich) for 30 min at 30 °C.

The resulting NPG films were then transferred onto polished glassy carbon electrodes

(GCEs, diameter: 4 mm). Cyclic voltammetry (CV) of the NPG electrodes was performed

in 1 M H2SO4 (-0.2-1.65 V vs. SCE, scan rate of 100 mV s-1 for 15 scans) to clean the

NPG surface, after which the electrodes were washed with water and left to dry in air

prior to further modification.

Enzyme and drug immobilisation

A 5.3 μl aliquot of a 6 mg ml-1 aqueous suspension of Os(bpy)2PVI, 1.3 μl of a 15 mg ml-

1 aqueous solution of PEGDGE and either 3.2 μl of a 10 mg ml-1 solution of GOx or BOx

were homogenously mixed using a vortex mixer. Solutions of the enzyme/polymer

mixture were drop cast on to NPG electrodes to prepare the NPG/Os(bpy)2PVI-GOx and

NPG/Os(bpy)2PVI-BOx bioelectrodes, respectively. The electrodes were immediately

placed in a vacuum desiccator that was connected to a vacuum pump for 10 min. The

electrodes were then transferred to a refrigerator (4oC) and allowed to dry overnight in

the dark.

A second layer of PEDOT doped with Amp was subsequently electrodeposited onto the

NPG/Os(bpy)2PVI-BOx biocathode, resulting in NPG/Os(bpy)2PVI-BOx/PEDOT-Amp.

To achieve this, the NPG/Os(bpy)2PVI-BOx biocathode was immersed into a solution

containing 0.1 M pH 7.0 PBS with 2 mM polyethylene glycol 3400 (PEG3400), 20 mM

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EDOT and 10 mM Amp and a 60 s pulse sequence at 0.9 V (2 s) and -0.4 V (3 s) was

applied. The electrodes were then gently rinsed with PBS for 30 min to remove loosely

bound Amp.

Material characterisation

Scanning electron microscopy (SEM, Hitachi SU-70, 15 kV) and transmission electron

microscopy (TEM, Tecnai G2 T20, 200 kV) were used to characterise the electrode

surface. The average pore size of NPG and deposition layer thickness were each

measured at least 30 times with ImageJ software (National Institutes of Health, Bethesda,

Maryland) [5].

Fourier transform infrared (FTIR) analysis of the various polymeric layers, which were

coated on Au foils (thickness: 0.1 mm, purity: 99.9%) using the same procedures for

NPG modification, was conducted on a Bruker Alpha FTIR spectrometer.

Electrochemical measurements

Electrochemical characterisation was carried out using a CHI802 potentiostat (CH

Instruments, Austin, Texas) with a three-electrode system consisting of NPG based

working electrodes, a saturated calomel electrode (SCE) reference electrode and a

platinum counter electrode. All experiments were performed at room temperature (20

±2°C) unless stated otherwise. Maximum background corrected catalytic current density

(jmax) for bioelectrodes was obtained by subtracting the background current density of the

electrode in a blank solution from that in substrate-containing solution.

The assembled EBFCs were characterised in a two-electrode configuration using a

NPG/Os(bpy)2PVI-GOx bioanode as the working electrode and a NPG/Os(bpy)2PVI-

BOx/PEDOT-Amp biocathode as the combined counter/reference electrode. The current

flow in the potential range between the open circuit voltage (OCV) of the EBFC and 0 V

was recorded at a scan rate of 1 mV s-1. Power density profiles were calculated using the

current output as a function of potential.

Drug release studies

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The release of Amp from an EBFC consisting of a NPG/Os(bpy)2PVI-GOx bioanode and

a NPG/Os(bpy)2PVI-BOx/PEDOT-Amp biocathode was carried out under open circuit

mode (spontaneous release) or EBFC-triggered mode in a cell containing 2 mL of air-

equilibrated buffer (0.1 M, pH 7.0 PBS, 10 mM glucose). Cumulative Amp release in

solution was monitored using a Cary 60 UV-Vis spectrophotometer (Agilent, USA). A

calibration curve was obtained by plotting the absorbance at 254 nm versus the

concentration of Amp. The calculated amount (μmol cm-2) of Amp released in the

solution was normalised with respect to the geometric surface area of the biocathode.

Anti-bacterial testing

A culture of Escherichia coli Top10 was grown overnight in a shaking incubator at 37°C

and 150 rpm. The culture was then sub-cultured (1:100) into fresh LB broth

supplemented with 1% glucose for Amp release testing. Ex situ and in situ release of

Amp was carried out.

Ex situ release. An EBFC consisting of a NPG/Os(bpy)2PVI-GOx bioanode and a

NPG/Os(bpy)2PVI-BOx/PEDOT-Amp biocathode was incubated in a 2 mL LB broth

supplemented with 1% (ca. 92.5 mM) glucose and operated at 0.15 V for 20 min. The

sub-cultured (1:100) E. coli was cultured in 2 mL of LB with ex situ release of Amp

(positive control, a). Three controls, b) EBFC operated at open-circuit, c) growth control

with blank broth added to the media, and d) manual addition of 1 mg mL-1 Amp, were

also prepared.

In situ release. The sub-cultured (1:100) E. coli was incubated in fresh LB broth

supplemented with 1% glucose, and grown for 2 h. In situ release of Amp was performed

by operating the EBFC at 0.15 V for 20 min in the same media containing E. coli (a). The

same control groups (b-d) were also prepared. All tests and controls were supplemented

with 1% glucose (due to the fact that growing E. coli will preferentially use glucose).

Samples were then grown for another 5 hours.

For each sample, optical density measurements at a wavelength of 600 nm (OD600) were

taken using a 1:100 dilution (dilution were undertaken using sterile distilled H2O) of all

cultures on a Varian Cary 100 UV–Visible spectrophotometer every hour for 5 hours.

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Supplementary figures

Fig. S1. Mediated electron transfer route on the bioanode (A) and biocathode (B).

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Fig. S2. Structure of Os(bpy)2PVI (A), PEDOT (B) and Amp (C).

Fig. S3. Linear scan voltammograms (LSVs) of the NPG/Os(bpy)2PVI-GOx bioanode (A)

and the NPG/Os(bpy)2PVI-BOx biocathode (B) in the presence of 0.2 mM Amp; Scan

rate of 2 mV s-1.

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Fig. S4. FTIR spectra of Au/Os(bpy)2PVI-BOx and Au/Os(bpy)2PVI-BOx/PEDOT-Amp.

Fig. S5. SEM (A-C) and TEM (D-F) images of bare NPG (A, D), NPG/Os(bpy)2PVI-

BOx (B, E) and NPG/Os(bpy)2PVI-BOx/PEDOT-Amp (C, F).

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Fig. S6. Potential spectrum for an EBFC using two different Os complex based polymers

to enable use of a higher OCV.

Fig. S7. Calibration curve showing linear relationship between the absorbance at 254 nm

and the concentration of Amp.

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Fig. S8. Current density profile of the cell comprised of a NPG/Os(bpy)2PVI-GOx

bioanode and a NPG/Os(bpy)2PVI /PEDOT-Amp “biocathode” without BOx in air-

equilibrated 0.1 M pH 7.0 PBS with 10 mM glucose.

Note: The short-circuit current density of such a control cell was only 0.21 µA cm-2, 1.6%

of an EBFC with a NPG/Os(bpy)2PVI-BOx /PEDOT-Amp biocathode (13.5 µA cm-2,

Fig. 1C). The OCV of the control cell (0.127 V) was also far less than the EBFC with

BOx (0.403 V, Fig. 1C). The control cell registered negligible power output and release

of Amp.

Fig. S9. Cyclic voltammograms of the NPG/Os(bpy)2PVI-GOx bioanode (A) and BOx

modified biocathodes (B) in air-equilibrated LB broth at a scan rate of 5 mV s-1. (C)

Power and current density profiles of the EBFC consisting of a NPG/Os(bpy)2PVI-GOx

bioanode and a NPG/Os(bpy)2PVI-BOx/PEDOT-Amp biocathode in air-equilibrated LB

broth supplemented with 1% glucose.

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Supplementary references[1] E.M. Kober, J.V. Caspar, B.P. Sullivan, T.J. Meyer, Synthetic Routes to New Polypyridyl Complexes of Osmium(Ii), Inorg. Chem., 27 (1988) 4587-4598.[2] R.J. Forster, J.G. Vos, Synthesis, Characterization, and Properties of a Series of Osmium- and Ruthenium-containing Metallopolymers, Macromolecules, 23 (1990) 4372-4377.[3] X. Xiao, P.Ó. Conghaile, D. Leech, R. Ludwig, E. Magner, A symmetric supercapacitor/biofuel cell hybrid device based on enzyme-modified nanoporous gold: An autonomous pulse generator, Biosens. Bioelectron., 90 (2017) 96-102.[4] X. Xiao, J. Ulstrup, H. Li, J. Zhang, P. Si, Nanoporous gold assembly of glucose oxidase for electrochemical biosensing, Electrochimica Acta, 130 (2014) 559-567.[5] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, NIH Image to Imagej: 25 Years of Image Analysis, Nat. Methods, 9 (2012) 671.

[1] E.M. Kober, J.V. Caspar, B.P. Sullivan, T.J. Meyer, Synthetic Routes to New Polypyridyl Complexes of Osmium(Ii), Inorg. Chem., 27 (1988) 4587-4598.[2] R.J. Forster, J.G. Vos, Synthesis, Characterization, and Properties of a Series of Osmium- and Ruthenium-containing Metallopolymers, Macromolecules, 23 (1990) 4372-4377.[3] X. Xiao, P.Ó. Conghaile, D. Leech, R. Ludwig, E. Magner, A symmetric supercapacitor/biofuel cell hybrid device based on enzyme-modified nanoporous gold: An autonomous pulse generator, Biosens. Bioelectron., 90 (2017) 96-102.[4] X. Xiao, J. Ulstrup, H. Li, J. Zhang, P. Si, Nanoporous gold assembly of glucose oxidase for electrochemical biosensing, Electrochimica Acta, 130 (2014) 559-567.[5] C.A. Schneider, W.S. Rasband, K.W. Eliceiri, J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, NIH Image to Imagej: 25 Years of Image Analysis, Nat. Methods, 9 (2012) 671.

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