Bioelectrocatalytic reduction of oxygen at gold nanoparticles modified with laccase Vida Krikstolaityte, Alejandro Barrantes, Arunas Ramanavicius, Thomas Arnebrant, Sergey Shleev, Tautgirdas Ruzgas PII: DOI: Reference:

S1567-5394(13)00085-6 doi: 10.1016/j.bioelechem.2013.09.004 BIOJEC 6691

To appear in:

Bioelectrochemistry

Received date: Revised date: Accepted date:

26 April 2013 2 September 2013 9 September 2013

Please cite this article as: Vida Krikstolaityte, Alejandro Barrantes, Arunas Ramanavicius, Thomas Arnebrant, Sergey Shleev, Tautgirdas Ruzgas, Bioelectrocatalytic reduction of oxygen at gold nanoparticles modified with laccase, Bioelectrochemistry (2013), doi: 10.1016/j.bioelechem.2013.09.004

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ACCEPTED MANUSCRIPT Bioelectrocatalytic reduction of oxygen at gold nanoparticles modified with laccase Barrantesb,

b

Arunas

b

Arnebrant , Sergey Shleev , Tautgirdas Ruzgas *

Department of Physical Chemistry, Faculty of Chemistry, Vilnius University, Naugarduko

24, 03225 Vilnius, Lithuania;

Department of Biomedical Sciences, Faculty of Health and Society, Malmö University,

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b

Thomas

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a

Ramanaviciusa,

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b

Alejandro

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Krikstolaitytea,

Vida

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20506 Malmö, Sweden.

*Corresponding author. Tel.: +46 702349580; fax: +46 406658100.

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E-mail address: [email protected] (T.Ruzgas).

Abstract

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To characterise bioelectrocatalytic oxygen reduction at gold nanoparticles (AuNPs) modified with Trametes hirsuta laccase (ThLc) combined electrochemical and quartz crystal

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microbalance measurements have been used. The electrodes with different degree of AuNPmonolayer coverage, , have been studied. In every case of  close to theoretically possible 44 ThLc molecules adsorbed at 22 nm diameter AuNP. The bioelectrocatalytic current was recalculated down to the current at a single AuNP. Unexpectedly, the current at a single AuNP was higher when  was higher. The maximum current reached at a single AuNP was 31.10-18 A which corresponds to the enzyme turnover (kcat) 13 s-1. This rate is lower than the homogeneous ThLc turnover (190 s-1) suggesting partial denaturation of ThLc upon adsorption or that some ThLc are not in DET contact with the electrode surface.

Keywords: bioelectrocatalytic oxygen reduction, gold nanoparticle, laccase, direct electron transfer

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ACCEPTED MANUSCRIPT 1. Introduction Since the discovery of direct electron transfer (DET) between the redox enzymes and electrodes [1] it has been intensively studied [2-4] and applied to design biosensors [5, 6] and

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biofuel cells (BFCs) [7-12]. It has been found that the majority of enzymes show slow DET

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which limits their application for the development of the above mentioned biodevices or restricts the efficiency and stability of the device. A number of studies have proved that DET of redox enzymes could be improved by exploiting gold nanoparticles (AuNPs) [13-17]. The

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observed enhancement of DET can be simplistically explained by the fact that nanoparticle (NP) comes into close contact with the active centre of an enzyme and functions as an

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intermediate electron hopping site in the heterogeneous ET pathway [18]. We have recently demonstrated [17] that AuNPs enable DET based bioelectrocatalytic oxygen reduction

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(Fig. 1) by the laccase from basidiomycete Trametes hirsuta.

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Fig. 1

The laccase (Lc) is a blue multicopper oxidase with the active centre comprised of four

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copper ions classified as T1, T2 and T3 [19]. During DET based oxygen bioelectroreduction the electrons are transferred from the AuNP modified electrode surface to the T1 copper site

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and further onto the T2/T3 copper cluster where the oxygen is reduced to water (Fig. 1). It should be noted that the mechanism of DET for this enzyme adsorbed at carbon based electrodes is relatively well understood [2], while it is still debatable in case of metal electrodes, e.g., gold [20, 21]. To understand and implement bioelectrochemical reduction of oxygen at Lc modified electrodes is of high practical interest for designing BFCs [9, 22]. BFC is a device which converts the energy conserved in organic and inorganic materials into electrical energy with the help of enzymes or living cells [23, 24]. In this context one of the obvious aims is to develop high current density enzymatic FCs based on DET reactions. This aim can be reached by loading a high amount of redox enzymes into electrically conducting three-dimensional (3D) nanomaterials [16, 25, 26]. Examples of BFC electrodes based on DET of Lcs [27], bilirubin oxidases [16], peroxidases [28], fructose dehydrogenases [29], etc., have been demonstrated in numerous studies. Colloidal AuNPs are often used in BFC design [30, 31], however, other 3D gold nanostructures [28, 32] have also been investigated. Although BFC cathodes based on 3D AuNP assembly function relatively well [11, 16, 30], DET between the redox enzyme and the AuNP in these structures is difficult to characterise.

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ACCEPTED MANUSCRIPT The knowledge is important, e.g., for miniaturising BFCs. Thus, DET studies of the laccase from Trametes hirsuta (ThLc) at AuNPs have been addressed in this work, especially, being aware that stable and high current density providing 3D biocathodes can be assembled by

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using Lc and AuNP building blocks. The purpose of the present investigation was to answer

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the questions: (i) How many ThLc molecules adsorb at a single AuNP? (ii) What is an approximate current magnitude of oxygen bioelectroreduction at a single AuNP modified with ThLc? (iii) What is the rate of the limiting reaction which defines oxygen

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bioelectroreduction at the AuNP-ThLc structure? To answer these questions we have conducted combined quartz crystal microbalance with dissipation (QCM-D) and

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electrochemical study of AuNPs modified by ThLc.

2.1. Chemicals and materials. Na2HPO4.2H20,

KCl,

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2. Experimental

NaCl,

Na2SO4,

HAuCl4.3H20,

2,2‘-azino-bis(3-

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ethylbenzothiazoline- 6-sulphonic acid) diammonium salt (ABTS) and citric acid monohydrate were purchased from Sigma (St. Louis, MO, USA). Trisodium citrate-2-hydrate

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and NaF was obtained from Merck (Darmstadt, Germany). All chemicals were of analytical grade. Poly-L-lysine (PLL) with the molecular weight of 70-150 kDa (for calculations the

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average MW of 110 kDa was taken), 0.01 % w/v in water, was received from Sigma-Aldrich (Irvine, Ayrshire, UK). Buffers and all other solutions were prepared using deionized water (18.2 MΩ.cm) purified with PURELAB UHQ II system from ELGA Labwater (High Wycombe, UK).

The dispersion of spherical AuNPs with an average NP diameter of 22±4 nm was prepared by reducing HAuCl4 with sodium citrate [33] and dialyzing against water. Their mean diameter and standard deviation was determined by recording NP diameter distribution using atomic force microscopy after drying their dispersion on mica surface pretreated with PLL. The dispersion was stored in glass bottles at +4 °C when not in use. As verified by SDS/PAGE a homogeneous preparation of fungal Lc (EC 1.10.3.2) from basidiomycete Trametes hirsuta with molecular mass of 70 kDa (glycosylation 12 %, pI 4.2) was used [34]. The stock solution of ThLc with 14.4 mg/ml enzyme concentration in 0.1 M phosphate buffer, pH 6.5, was stored frozen at -18 °C. For the experiments the stock solution of the enzyme was thawed at +4°C and diluted to 0.7 mg/ml concentration. The ThLc activity and electrochemical measurements were performed in 50 mM phosphate buffer (pH 4.0)

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ACCEPTED MANUSCRIPT containing 0.1 M Na2SO4 (adjusted by citric acid). The appearance of a coloured product during the oxidation of ABTS catalysed by ThLc was exploited to evaluate the activity (turnover number) of ThLc [35]. The homogeneous turnover (kcat) of the enzyme was 190 s−1

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at room temperature. AT-cut gold-coated quartz crystals (QSX 301) with a fundamental

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frequency of 4.95 MHz were purchased from Q-Sense (Gothenburg, Sweden). The geometric area of the gold surface was 0.785 cm2, which was similar to the electrode area determined by

2.2. QCM-D and electrochemical measurements.

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cyclic voltammetry experiments (see supplementary materials).

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To quantify AuNPs and ThLc molecules attached to the surface, and to evaluate the bioelectrocatalytic properties of the AuNP-Lc layer on a planar gold electrode, simultaneous

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electrochemical and QCM-D measurements were performed using the QWEM401 electrochemistry cell (Q-Sense, Gothenburg, Sweden). All measurements were made at +22±0.02 °C. The planar surface of gold covered QCM-D sensor (AuQCM-D) was modified

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with PLL, AuNPs and the enzyme by the following procedure. PLL was adsorbed on the QCM-D sensor and then ThLc was deposited. The resulting structure was noted as AuQCM-D-

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PLL-ThLc. Afterwards, the AuNP layer was adsorbed following by adsorption of ThLc (AuQCM-D-PLL-ThLc-AuNPs-ThLc). We assumed that this final ThLc adsorption step resulted

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into the enzyme adsorption only on AuNPs since adsorption of ThLc directly on PLL covered gold sensor was already blocked by the first ThLc adsorption step. The conditions for each adsorption step as well as the calculation of adsorbed mass are described in supplementary materials.

After each adsorption step, i.e., for QCM-D electrode structure denoted as AuQCM-DPLL-ThLc, AuQCM-D-PLL-ThLc-AuNPs, and AuQCM-D-PLL-ThLc-AuNPs-ThLc, the electrode was assessed by linear sweep voltammetry (LSV) at 1 mV/s potential scan rate using compactstat from IVIUM (Eindhoven, Netherlands). Ag/AgCl/3 M KCl inserted into the outlet of the QCM-D flow cell served as a reference and a sputtered ring of platinum on the glass window of the cell as a counter electrode, respectively. The volume above the sensor was about 100 μl. LSVs were recorded under the flow (100 µL/min) of the buffer solution in the QCM-D cell. 3. Results and discussion To characterise ThLc interaction with AuNPs and assess bioelectrocatalytic properties of the AuNP-ThLc nanocomponent we carried out simultaneous QCM-D and LSV

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ACCEPTED MANUSCRIPT measurements. AuNPs were assembled on a planar gold surface of the QCM-D sensor, AuQCM-D, with subsequent adsorptive modification by ThLc. From our previous investigations we know that such an assembly enables considerable, DET based, bioelectrocatalytic

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reduction of oxygen (Fig. 1) at applied potential higher than the formal potential of the T1

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copper of ThLc [17]. However, as a number of ThLc molecules adsorbing and generating the bioelectrocatalytic current at a single AuNP are unknown it is difficult to understand the limitations and improvement possibilities of bioelectrocatalysis at the AuNP-ThLc

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assemblies. The mentioned characteristics, at least to some degree, could be assessed by using methods applied to measure surface bound mass (e.g., QCM-D) and the reduction

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current simultaneously.

The mass was monitored for surface structures obtained by the following adsorption

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sequence: AuQCM-D-PLL (PLL adsorbed on the QCM-D sensor surface), AuQCM-D-PLL-ThLc (PLL adsorption was followed by ThLc adsorption ), AuQCM-D-PLL-ThLc-AuNPs (AuNPs were adsorbed on PLL-ThLc treated QCM-D sensor surface), and AuQCM-D-PLL-ThLc-

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AuNPs-ThLc (ThLc was adsorbed on surface modified by PLL-ThLc-AuNPs). We should mention that notations of surface structures, e.g., AuQCM-D-PLL-ThLc-AuNPs-ThLc, reflect

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rather the sequence of the surface modification than the real layer structure, which is obviously more disordered. It was experimentally found that AuNPs did not adsorb on the

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ThLc modified planar AuQCM-Delectrode if the modification with PLL was omitted. These experiments revealed that AuNP assembly is due to the AuNP interaction with PLL layer. The PLL-AuNP interaction was also negligibly affected by the ThLc layer adsorbed on AuQCM-D-PLL surface structure since there was no noticeable difference in the amount of adsorbed AuNPs on AuQCM-D-PLL and AuQCM-D-PLL-ThLc surface. This indicates that the majority of ThLc molecules adsorb on PLL non-covered planar AuQCM-D sensor surface. Due to the importance of PLL for the assembly of the AuNP-ThLc layer, PLL adsorption is discussed in supplementary materials concluding that the amount of adsorbed PLL on AuQCMD

surface was equal to 0.8±0.2 pmol/cm2.

3.1 Sequential adsorption of Lc, AuNPs and Lc After the PLL layer was adsorbed on AuQCM-D sensor and rinsed with water the ThLc solution was pumped through the QCM-D flow cell. QCM-D measurements revealed that ThLc adsorbed on top of PLL treated Au surface. The surface concentration of the enzyme calculated using the Voigt model was equal to 3.5±0.8 pmol/cm2 which was much higher than the concentration of adsorbed PLL (0.8±0.2 pmol/cm2, see supplementary material).

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ACCEPTED MANUSCRIPT Higher adsorption of the enzyme is reasonable because ThLc is a globular protein compared to the coil-shaped PLL. ThLc is slightly net positively charged (pI = 4.2) at pH 4 (in the adsorption conditions). The electrostatic PLL-ThLc interaction is not expected at this pH.

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Locally, it still might take place between negatively charged areas of the protein and PLL,

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especially, at higher ionic strength. However, the majority of ThLc probably adsorbs at PLL non-occupied surface. The concentration of adsorbed ThLc is comparable to the concentration (2.6 pmol/cm2) of BSA on gold surfaces as previously measured by

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ellipsometry [36].

After the AuQCM-D sensor surface was pre-treated with PLL and ThLc, the layer of

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AuNPs was assembled on the sensor surface. The adsorption sequence is denoted by abbreviation AuQCM-D-PLL-ThLc-AuNPs. Experimental conditions were chosen to assemble

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the AuNP layer with NP packing density lower than monolayer, i.e., diluted monolayer. This was done by adsorbing AuNPs from NP dispersion with the same NP concentration, but with a different NaCl concentration. Our experiments showed (Fig. 2) that the degree of

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monolayer coverage,  (Eq. 1), depended on the NaCl concentration present in the AuNP dispersion.  is defined as the ratio of experimentally determined surface concentration of

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NPs, Ca, to their surface concentration maximally possible by hexagonal packing, Ca,max. For 22 nm diameter AuNPs Ca,max is estimated to be equal to 0.396 pmol/cm2.

Fig. 2

Ca Ca ,max

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

(1)

It is hard to believe that the adsorption sequence PLL-ThLc-AuNPs results into an idealised layered structure as can be interpreted from the abbreviation AuQCM-D-PLL-ThLcAuNPs. However, the appraisal of the AuQCM-D-PLL-ThLc-AuNP surface structure is very important for later interpretation of bioelectrocatalysis down to a single AuNP level. This is why we analysed the dependence of  on the concentration of NaCl (Fig. 2) in the light of idealised AuNP adsorption model which considers electrostatic repulsion between the AuNPs as the major factors determining surface density of NPs [37]. It should be noted that more comprehensive theoretical models, additionally accounting for van der Waals inter-particle interactions [38] or double layer effect of planar surface [39], were not used in the

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ACCEPTED MANUSCRIPT rationalisation of our data because the evaluation of these interactions is beyond the scope of this study. The dependence  vs. dimensionless multiple of ·rNP shown in Fig. 2 can be

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modelled by Eq. 2 (Fig. 2, curve b), where rNP is a geometric (“hard”) radius of NPs and  is

2

2

2 F 2 zi2 ci  0 RT

(2)

(3)

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

2

     rNP    jam  rNP      jam   r      r     1    NP   NP  

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 r    jam  NP  rNP,eff

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a reciprocal of Debye length (Eq. 3).

F and R denote Faraday and Molar gas constants, respectively; zi and ci denote the valency and the concentration of electrolyte ions;  and 0 denote the dielectric constant of

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the solution and vacuum, respectively; and T stands for absolute temperature. In Eq. 2 jam is a jamming limit of  for a random sequential adsorption of NPs

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experimentally determined to be equal to 0.547, i.e., jam = 0.547 [37]. Effective (“soft”) radius, rNP,eff, of nanoparticles (Eq. 2) accounts for the repulsive forces between the NPs

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resulting into a diluted NP monolayer coverage, i.e., for  < jam. In case of electrostatic repulsion of NPs the effective radius can be estimated as a multiple of Debye screening

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length, -1 (Eq. 2). Parameter , to the highest degree, accounts for the effect of electrical surface potential of NPs on  [40]. However,  depends also on  and rNP [39] and obviously includes all interactions that are included into theoretical modelling of inter-particle interaction potential at the interface. Kooij [41], however, demonstrated that accounting only for particle-particle electrostatic repulsion satisfactory describes  for diluted NP monolayer. Following mathematical formalism justified by Kooij [41]  values were calculated to increase from  = 1.05 (at 0.1 mM NaCl) to  = 1.68 (at 20 mM NaCl) for 22 nm diameter NPs (rNP = 11 nm) with the surface potential of 0.05 V. Mathematical formulas used in the calculation are summarised in the supplementary materials including full set of  values. By substituting these  values in Eq. 2 a theoretical  vs. ·rNP dependence is drawn in Fig. 2 (curve b). From Fig. 2 it can be seen that AuNP assembly from dispersion dialyzed against water (concentration of NaCl is 0 mM) results in a surface concentration corresponding to

 = 0.33. Increasing NaCl concentration in AuNP dispersion up to 20 mM obviously diminishes the repulsion between AuNPs allowing higher AuNP packing density on the

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ACCEPTED MANUSCRIPT surface. A nearly complete, i.e.,  = 0.8, AuNP monolayer coverage was reached using the AuNP dispersion containing 10 mM NaCl. While in the presence of 20 mM NaCl the estimated surface coverage approached  = 1 (Fig. 2). It should be noted that AuNP

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dispersion containing 20 mM NaCl was not stable for longer than 30 min and hence the onset

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of multilayer formation might be expected.

From Fig. 2 it can be seen that the theoretical dependence of  on the dimensionless product ·rNP (Fig. 2, curve b) describes the experimental points rather poorly. This indicates

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that the model of random sequential adsorption with jamming of NPs does not explain AuNP adsorption on the PLL treated surface. The model of random adsorption with jamming of NPs

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seems to suit relatively well for the surfaces modified with the compounds of small molecular weight, e.g., silanes [39, 41] or thiols [42]. However, it does not explain AuNP adsorption on

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PLL modified surfaces. Similar conclusion was drawn in an earlier study of ferritin adsorption on PLL modified polystyrene [37]. The authors found more than monolayer coverage of ferritin on the PLL treated surface. Taking into account previously published

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results and our data presented in Fig. 2 we can assume that certain parts of long PLL molecules probably detach from the planar gold surface and wrap AuNPs. This phenomenon

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is highly likely to enable the higher surface density of AuNP as it is allowed by random sequential adsorption model. Pointing that PLL can wrap AuNPs becomes important when

below.

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the explanation of bioelectrocatalysis per single AuNP modified with ThLc is discussed

Assembly of bioelectrocatalytic system was completed by adsorbing the second layer of ThLc. The final structure is denoted as AuQCM-D-PLL-ThLc-AuNPs-ThLc. The surface concentration of the last ThLc layer was dependent on the coverage of AuNPs. Specifically, the estimated surface concentrations were 9±1, 8.9±1.9, 9.9±1.6, 14±4, and 24±5 pmol/cm2 (geometric sensor area, i.e., AuQCM-D) at AuNP coverage  of 0.33, 0.35, 0.49, 0.81, and 1, respectively. It can be noted that adsorption of ThLc bound on the AuNP surface (at AuNP  of 1) was approximately 7 times higher if compared to the first ThLc layer adsorbed on AuQCM-D-PLL surface (3.5±0.8 pmol/cm2). It is important to emphasize that a part of the mass accounted for ThLc can come from the mass of coupled water sensed by QCM-D. A typical part of coupled water for different proteins can vary from 30 % to 90 % [43]. We have neglected the mass of coupled water (taken as 0 %) when assessing the number of ThLc molecules adsorbed per single AuNP (Fig. 3). From Fig. 3, especially at  > 0.4, it can be concluded that experimentally found number of ThLc molecules adsorbed per single AuNP is

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ACCEPTED MANUSCRIPT close to the theoretical estimate of 44 laccase molecules per 22 nm diameter NP. Reasonably, this number is practically independent on AuNP monolayer coverage. The theoretical ThLc number per AuNP was calculated assuming that each ThLc molecule (PDB database: 3FPX)

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occupies a 6.4x5.4 nm2 rectangle.

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Fig. 3

3.2 Oxygen bioelectrocatalysis

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DET characteristics of ThLc enzyme at AuNPs were investigated by running LSVs after each modification step of AuQCM-D electrode. Fig. 4 shows LSVs recorded for the case of

bioelectrocatalytic oxygen reduction.

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Fig. 4

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the AuNP layer with  = 1, i.e., the electrode which generated the highest current of

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From Fig. 4 it can be seen that decreasing the applied potential causes the increase of the reduction current starting at approximately 650 mV and reaching the maximum at

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400 mV. It is well established that the current is due to the bioelectrocatalytic reduction of oxygen at the ThLc-AuNP modified electrode [17]. Inhibition of laccase by NaF (Fig. 4, dotted curve) entirely diminishes the current. Practically, no bioelectrocatalytic current was observed, if the electrode had been modified only by ThLc (structure AuQCM-D-PLL-ThLc, Fig. 4, grey curve) or AuNPs (structure AuQCM-D-PLL-ThLc-AuNPs, Fig. 4, solid curve). The bioelectroreduction current was higher at a higher AuNP monolayer coverage (Fig. 5) as expected due to a higher amount of AuNPs modified with ThLc molecules at the electrode.

Fig. 5

Dividing current density (Fig. 5a) by a known amount of AuNPs at the electrode, the bioelectroreduction current at a single AuNP was calculated (Fig. 5b) which reached the maximum of 31.10-18 A. Surprisingly, the current per AuNP modified with ThLc molecules depended on the NP density, or , on the electrode surface. To explain this dependence (Fig. 5b) we considered three limiting steps of bioelectrocatalysis, which determine the bioelectrocatalytic current, i, at the electrode:

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ACCEPTED MANUSCRIPT 1 1 1 1    i iDET ibio imt

(4)

where iDET, ibio, and imt, are electrode current which is limited by DET between the ThLc and

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AuNP, catalytic activity of ThLc, and mass transfer of oxygen, respectively. The limitation of

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the electrode current by the diffusion of oxygen (imt) can be easily excluded from the consideration as experimentally increasing the rate of solution flow at the electrode has not increased the electrode current. The kinetic currents, iDET and ibio, can be described by the

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kinetic scheme presented in Fig. 1, i.e., adopting the approach elaborated by Climent et al [44]. The equations defining the currents are provided below and the final formulas used for

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fitting the experimental results are presented in the supplementary information.

k2  k0 exp[(1   )

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F ( E  E T01' )] RT F ( E  ET01' )] RT

ibio  nFAk5  nFA

kcat [O2 ] K M  [O2 ]

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k1  k0 exp[ 

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iDET  FA[k1 (1  P1 )  k2 P1 )]

(5) (5a) (5b) (6)

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In Eqs. 5-6 F is Faraday constant, A is a geometric area of the electrode,  is surface concentration of AuNP-bound ThLc molecules, k1 and k2 are potential dependent DET rate constants defined by the Butler-Volmer formalism (Eqs. 5a and 5b). P1 represents a fraction of NP-bound ThLc molecules with the reduced, Cu(I), T1 copper centre. k0 is the standard heterogeneous ET rate constant, R is the gas constant, T is temperature, E is applied potential, ET01' is the formal redox potential of the T1 copper centre,  is the transfer coefficient. n= 4 is

a number of electrons of the reaction (see Fig. 1), k cat is a homogeneous catalytic rate constant (turnover) of the enzyme, [O2] is oxygen concentration (substrate) at the electrode surface and KM is a Michaelis-Menten constant. Fitting the experimental bioelectrocatalytic currents of the electrodes characterised by different  of AuNPs (e.g., Fig. 4 for  = 1) to the Eqs. 5-6 kcat and k0 constants were assessed. The calculation results are presented in Table 1.

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ACCEPTED MANUSCRIPT Table 1. Kinetic constants, kcat and k0, characterising bioelectrocatalytic reduction of oxygen at electrodes modified with ThLc and AuNPs. The constants were calculated using Eqs. 5-6. Parameters  = 0.5; KM = 100 µM; and [O2] = 250 µM were fixed. Columns summarise the

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following: [NaCl] - sodium chloride concentration in water during the assembly of AuNPs, 

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- degree of monolayer coverage by AuNPs,  - surface concentration of ThLc on AuNPs, ET10’ - the formal potential of the T1 copper site estimated by fitting procedure

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[NaCl], mM

k0, s-1

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(supplementary materials).

1.40

0.38

0.63

1.41

0.39

0.64

0.99

2.75

0.55

0.66

0.81

0.89

9.10

4.2

0.66

1

0.91

13.0

6.1

0.66



, pmol/cm2

0.33

2.37

1

0.35

1.4

5

0.49

10 20

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0

kcat, s-1

ET10’ vs. Ag/AgCl, V

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As can be seen from Table 1, kcat and k0 increase by approximately 10 and 16 times when  increases from 0.33 to 1. This apparently means that the enzyme becomes more active at

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higher  as well as DET coupling improves considerably when the AuNP density on the surface increases. The dependence of the constants on  cannot be explained considering that ThLc-AuNP interaction remains the same at a different AuNP density on the electrode surface. For reasoning the dependence of the constants on , we can put forward several hypotheses. The first, yet hardly probable, is that an unknown impurity occupies the surface of AuNPs, when NP density on the surface is low. The second hypothesis is that PLL probably partly lifts from the planar AuQCM-D surface and wraps up the surface of AuNPs impairing proper orientation of ThLc on AuNPs. PLL acts as an “impurity” limiting the DET coupling between the enzyme and AuNP as well as the amount of ThLc in DET contact, though similar ThLc amount adsorbs per AuNP at different  (Fig. 3). As noted in §3.1, this explanation is also in line with the observation of much higher AuNP coverage than jamming limit, which is practically possible if PLL partly wraps AuNPs bound at the planar AuQCM-DPLL surface. As can be seen from Table 1 the maximal values of kcat = 13 s-1 and k0 = 6.1 s-1 were obtained in conditions when AuNP assembly was carried out from the NP dispersion containing 20 mM NaCl ( = 1). The constants can probably be underestimated since the

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ACCEPTED MANUSCRIPT mass of coupled water measured by QCM-D cannot be excluded from the determined mass of the ThLc layer. If we accept that about 50 % of the mass in the ThLc layer comes from the coupled water, then the estimated kcat could be twice as high. Nevertheless, comparing this to

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the kcat (190 s-1) known from the homogeneous activity assays, it can be concluded that DET

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coupling of ThLc to AuNP by simple adsorption still could be improved. To deeper understand DET of laccases at AuNPs we continue with the measurements of the dependence

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of oxygen bioelectrocatalysis on the size (radius) of AuNPs.

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4. Conclusions

In this work we assembled the AuNP layer on a planar QCM-D gold electrode (QCMD sensor). The density of the surface bound AuNPs was regulated by NaCl concentration in

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the AuNP dispersion. The AuNP monolayer coverage () ranged from of 0.33 to 1. A Poly-Llysine (PLL) at the planar surface of a gold QCM-D sensor was essential for the assembly of

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AuNPs. Surface bound AuNPs were modified with Trametes hirsuta laccase (ThLc). From combined QCM-D and electrochemical measurements the maximum bioelectrocatalytic current of oxygen reduction of 31.10-18 A at a single AuNP (diameter of 22 nm) modified

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with ThLc was estimated. This current was obtained at  = 1 of AuNP monolayer coverage. At a lower surface coverage of AuNPs the current at a single AuNP was lower. This was

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explained by PLL wrapping AuNPs and impairing DET wiring of the enzyme to nanoparticle. In this situation not all adsorbing ThLc molecules are electronically connected to AuNP, which formally results in kcat (13 s-1) for ThLc molecules at AuNP being much lower than kcat (190 s-1) determined for the enzyme in homogeneous activity assays.

Acknowledgement Authors thank for financial support: TR and SS - the Swedish Research Council and the European Commission, VK - the Lithuanian Research Council and the Swedish Institute, TA - Th. Ohlsson foundation.

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ACCEPTED MANUSCRIPT References

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ACCEPTED MANUSCRIPT Figure Captions Fig. 1 The schematic representation of DET between a Lc molecule (green ribbons) and the AuNP modified planar gold electrode. The T1 copper site and the T2/T3 copper cluster in Lc

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are presented as blue dots. Red arrows represent the direction of ET and the reaction of

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oxygen reduction to water at the applied potential below 650 mV (vs. Ag/AgCl/3M KCl). The scheme represents a sketch summarising the mechanism of bioelectroreduction of oxygen at

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the electrode as described by Eqs. 4-6.

Fig. 2 The degree of monolayer coverage () by 22 nm diameter AuNPs: (a) experimental

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and (b) theoretical  dependencies vs. the product of the Debye screening length (-1) and NP radius (rNP).  = 1 corresponds to complete 2D hexagonal packing. Theoretical dependence

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(b) is calculated for random sequential adsorption model, Eq. 2.  values are presented in Table S1, supplementary materials. Arrows mark the concentration of NaCl in AuNP

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dispersion used during AuNP assembly.

Fig. 3 The number of ThLc molecules r(ThLc) adsorbed per one AuNP at a different AuNP

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monolayer coverage. The solid line shows 44 theoretically expected ThLc molecules per one

Fig. 4 LSVs obtained using the AuQCM-D electrode modified with the PLL-ThLc-AuNP-ThLc biocatalytic structure. The assembly of AuNP was made from 20 mM NaCl (resulting  = 1). Potential scan rate: 1 mV/s; flow rate: 100 μL/min; 50 mM citrate - phosphate buffer, pH 4.0. Fig. 5 The dependence of bioelectroreduction of oxygen on AuNP monolayer coverage (): (a) current density per geometric electrode area (AuQCM-D); (b) the absolute current at a single AuNP. The values of the bioelectrocatalytic current are taken at E = 400 mV vs. Ag/AgCl using the data of LSV experiments (See Fig. 4 for  = 1). The geometric electrode area was 0.785 cm2. Current per NP was evaluated dividing the measured current density by the AuNP number at cm2 estimated by QCM-D measurements.

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Oxygen electroreduction at laccase modified gold nanoparticles (AuNP) was studied QCM-D and electrochemistry were combined Bioelectrochemical oxygen reduction at single AuNP was assessed The current at single AuNP increased at higher degree of their monolayer coverage

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