1

Direct electrochemistry of cyt c and hydrogen peroxide biosensing on

oleylamine- and citrate-stabilized gold nanostructures.

Ekaterina Koposovaa,b, Galina Shumilovab, Yury Ermolenkob, Alexandre Kisnerb,c, Andreas

Offenhäussera, and Yulia Mourzinaa

a Peter Grünberg Institute 8, Forschungszentrum Jülich GmbH, 52425 Jülich, and Jülich-

Aachen Research Alliance - Future Information Technology (JARA-FIT)

b Faculty of Chemistry, St. Petersburg State University, 199034 St. Petersburg, Russia

c current affiliation: Cell Biology and Neuroscience Department, Rutgers University, 604

Allison Road – Piscataway, NJ

Corresponding Author:

Dr. Yulia Mourzina

Peter Grünberg Institute 8, Forschungszentrum Jülich GmbH, 52425, Jülich and Jülich-

Aachen Research Alliance (JARA_FIT). Tel: +49 2461 612364; Fax: +49 2461 618733; E-

mail: y.mourzina@fz-juelich.de

Abstract. Two types of gold nanostructures are discussed: charge(citrate)-stabilized and

sterically(OA)-stabilized gold nanostructures and their assembly with heme-containing

metalloproteins, cyt c and horseradish peroxidase (HRP) enzyme into a bioelectrochemically

active nanoarchitecture as well as a sensor application. The assembly of the nanostructures on

the thin-film gold electrodes and the immobilization and electrochemical properties of

metalloproteins on these electrodes are presented. The nanostructured bioelectrochemical

interfaces provide a good environment for the stable and reproducible immobilization of

electroactive proteins. We show that both molecules preserve their functionalities

(electrochemical and biocatalytic activities). The amount of electroactive proteins

immobilized on the nanostructured electrode surfaces is significantly increased compared to

the flat electrode surfaces. The kinetic parameters of the heterogeneous direct electron transfer

reaction of cyt c on the nanostructured electrodes are compared. The thin-film gold electrodes

modified with OANWs, OANPs, and citrate-stabilized NPs and covalently immobilized HRP

exhibit an excellent catalytic activity towards the reduction of hydrogen peroxide with a

working concentration range from 20 µM to 500 µM, a sensitivity of 0.031 A M-1 cm-2 (RSD

2

0.005), 0.027 A M-1 cm-2 (RSD 0.004), and 0.022 A M-1 cm-2 (RSD 0.0035), and a detection

limit of 5 µM, 8 µM, and 14 µM, respectively (RSDs near the detection limits were 9 to 12

%). The HRP sensor characteristics are improved significantly compared to the flat thin-film

sensors by using gold nanostructures. Our study shows that ultrathin gold nanowires and

nanoparticles with two different types of stabilizing agents are promising materials for

assembling biomolecules into functional nanoarchitectures for metalloprotein-based

bioelectrochemical sensors.

Keywords: biosensor, gold nanowire, gold nanoparticle, metalloprotein, cytochrome c,

horseradish peroxidase

1. Introduction

Modern developments of (bio)chemical sensors explore nanostructured materials to

improve sensor characteristics. The nanostructures can improve the interface between the

biomolecules and an electronic transducer reducing the distance between the redox center of

proteins and an electrode for a direct electron transfer [1]. Three-dimensional nanostructured

materials provide a favorable surface for the immobilization of biomolecules allowing them to

retain their biological activity due to the enhanced orientation freedom, thus preventing

denaturation of biomolecules and encouraging longer stability and higher reproducibility of

the metalloprotein functions on the nanostructured electrodes [2]. Furthermore, geometrical

signal enhancement due to an increase of the effective electrode surface area by three-

dimensional nanostructures can be achieved using nanomaterials [3].

Due to their optical, electrical and chemical properties, noble metal nanostructures are

promising materials for applications in nanoelectronics, medicine, and catalysis. Various

methods are used to prepare stable gold nanostructures based on the principle of dispersion

and condensation, where in the latter method the metal nanoparticles are produced by

reducing the metals ions of the corresponding metal salts. In most cases, gold nanoparticles

are produced by reducing gold (I) or (III) with various organic and inorganic reducing

substances, which can additionally play the role of a stabilizing agent, or/and by

photoreduction [4,5]. In the pioneering studies of M. Faraday [6] and R. Zsigmondy [7],

formaldehyde, ethanol, and white phosphorus were used as reductants for preparing

nanoparticles with a diameter of 5 to 12 nm. Widely used methods are based on such reducing

and stabilizing agents as sodium citrate (producing gold colloids in water of around 10 to 20

nm in diameter) [8], ascorbic acid (12 nm) [9], EDTA (20 nm) [10], sodium borohydride

3

[4,11], oleic acid, and oleylamine [12-14], and “green syntheses” with non-toxic agents such

as fruit extracts, allowing nanostructures of various sizes and geometries to be prepared [15],

and glucose and starch as reducing and stabilizing agents, respectively [16]. Sodium or

potassium thiocyanate was used [17] to prepare ultra-dispersion nanoparticles (2-3 nm).

Anisotropic growth conditions should be created in order to prepare non-spherical

nanostructures with high aspect ratios [18]. Nanowires of approx. 2 nm diameter and up to

several µm in length have been prepared using OA as a stabilizing and triisopropylsilane as a

reducing agent [19] and only OA as both the stabilizing and reducing agent [12-14]. The

dimensions of the nanowires depend strongly on the molar ratio of the precursor salt,

stabilizing and reducing agents as well as on solvent polarity, pH, temperature, and presence

of substances promoting the anisotropic growth of nanowires [20-22]. Factors and

mechanisms promoting anisotropic growth and the formation of nanowires and other high

aspect ratio nanostructures have been reviewed [4,13,21,23-25]. Understanding the properties

and advantages of metal nanostructures in comparison to bulk metal encourages their

application in biosensors.

Investigations have focused on assembling nanoelectronic elements and biomolecules

with evolutionarily optimized functions, such as recognition, binding, carrier, and catalysis,

into bioelectronic systems [1,26-35] for sensors, bioelectrocatalysis, biofuel cells, and solar

energy conversion. Heterogeneous electron transfer reactions of redox proteins functioning,

for example, as electron transfer mediators of redox reaction chains and oxidoreductase

enzymes catalysts can be used for the development of bioelectrochemical sensors and

bioelectrocatalytical systems.

Metalloproteins, in particular, heme proteins, such as cytochrome c (cyt c), and

oxidoreductase enzyme horseradish peroxidase (HRP, EC 1.11.1.7.), are often used to study

the properties of bioelectronic interfaces because they provide stable and reliable systems and

are interesting for fundamental and applied studies. Cyt c is one of the most extensively

studied metalloproteins because of its central role in electron transfer in living organisms, and,

as a consequence, in the electron shuttle between molecular partners in artificial bioelectronic

systems and biofuel cells [36,37]. The conjugation of cyt c with nanoelectronic elements

[16,38,39] may provide bioelectronic systems with favorable properties with respect to the

electron transfer rate of cyt c ferri/ferro states and the amount of immobilized electroactive

metalloprotein. Horseradish peroxidase (HRP) is one of the most widely used enzymes in

analytical biochemistry for the construction of biosensors and for immunoassays. HRP-based

sensors are also used in a bi-enzymatic approach for the detection of hydrogen peroxide

4

produced in the reaction of a wide spectrum of oxidoreductase enzymes with their substrates

and for the bioelectrochemical monitoring of phenols, aminophenols, and other donor

substrates [40]. Optical and electrochemical methods are the most used methods for

determination of hydrogen peroxide. Electrochemical methods with sensors are attractive for

practical applications, because they are associated with small-size, low energy consumption,

low cost, and portability, when compared to many other analytical techniques.

The materials and surface of the electrode are of primary importance for an effective

bioelectrochemical interface. Various aspects are taken into account, such as the geometry

and morphology of the nanomaterials, immobilization of biomolecules on the electrode

surfaces, and electron transfer at the interfaces. Noble metals and carbon electrode materials

have been used most successfully [41]. On some of the electrode materials, particularly on

carbon electrodes, heterogeneous direct electron transfer has been reported between an

electrode and an electrocatalytic center of redox proteins [42-46]. Nevertheless, redox centers

of biomolecules generally lack electron transfer communication with electrode transducers

because of the insulating protein matrix. The rate of electron transfer between the electrode

and a redox center of proteins is negligibly small and drops by a factor of e for each 0.91 Å

[47-49]. Therefore, artificial redox groups or mediators have been used to mediate electron

transfer between redox centers of biomolecules and electrodes. Approaches to incorporating

these artificial mediators include covalent attachment of electron relays to biomolecules,

conductive polymers, and redox hydrogels, and soluble artificial redox mediators [50].

Electron transfer pathways in biosensors have been reviewed [32,50].

A great variety of nanomaterials and their geometries and modifications, numerous

analyzed substances as well as sensor types complicate the analysis of data on

bioelectrochemical nanosensor materials. Therefore, authors usually focus on one type of

nanomaterial or one type of geometry of the nanostructures employed in one particular sensor

type.

In this study, we compare two types of gold nanostructures: charge(citrate)-stabilized

and sterically(OA)-stabilized gold nanostructures as an electrode platform for heme-

containing metalloproteins, cyt c and HRP enzyme. Although, the synthesis of gold

nanostructures employing OA is versatile and inexpensive, the application of the

nanostructures prepared by this method has not been exploited for bioelectronic systems. One

of the reasons for this is that the surface of the nanostructures is insulated by stabilization

compounds, which preserve the integrity of nanostructures, but simultaneously form an

insulating barrier for the charge transfer reactions. It is thus challenging to obtain access to the

5

free or chemically functionalized surfaces for bioelectrochemical systems. We outline the

synthesis and assembly of gold nanostructures on the thin-film gold electrodes.

Immobilization and electrochemical properties of heme proteins cyt c and oxidoreductase

enzyme HRP on the electrodes modified with gold nanostructures are discussed. We show

that both molecules preserve their functionalities (electrochemical and biocatalytic activities).

The amount of electroactive proteins immobilized on the nanostructured electrode surfaces is

significantly increased compared to flat electrode surfaces. Kinetic parameters were obtained

for the direct electron transfer reaction of cyt c on the nanostructured electrodes. The HRP

sensor characteristics are improved significantly by using AuNSs. We demonstrate that both

types of gold nanostructures are favorable electrode materials for assembling metalloproteins

into a bioelectrochemically active nanoarchitecture as well as a sensor application for

designing biosensors, bioelectronic devices, and bioelectrocatalysis.

2. Experimental section

2.1. Reagents and materials. Benzenedithiol (BDT), cysteamine (Cys, 2-

aminoethanethiol), ethanedithiol (EDT), 6-mercaptohexanoic acid (6-MHA), 2-

mercaptoethanol (2-ME), glutaraldehyde solution (GA, 50 %, for electron microscopy),

triisopropylsilane, oleylamine, AuCl, HAuCl4.3H2O, cytochrome c (from horse heart, type

VI), and horseradish peroxidase (HRP, peroxidase from horseradish type VI-A) were

purchased from Sigma-Aldrich and used as received. Other chemicals were reagent grade.

Distilled water was used for the experiments.

2.2. Synthesis and structural characterization of gold nanostructures.

Citrate-stabilized gold nanoparticles (cit-NP) were prepared using the Turkevich

method [8] while stirring vigorously to produce monodisperse nanoparticles with a diameter

of 17 nm, where the citrate ions act both as reducing and capping agents. Oleylamine-

stabilized gold nanoparticles (OANP) were prepared according to [14]. Briefly, 0.05 g AuCl

was dissolved in a mixed solution composed of 2 mL of OA and 2 mL of hexane. The

solution was added to a glass vessel containing 18 mL of OA at 80 °C and the mixture was

left undisturbed for 5 to 6 h. The OANPs were precipitated by centrifugation, washed three

times with ethanol and finally redispersed in hexane. Oleylamine-stabilized gold nanowires

(OANW) were prepared using a modified procedure [19]: 7.45 mg HAuCl4.3H2O was mixed

with 200µL OA, 2.5 ml hexane was added, followed by 100 µL of TIPS. After a reaction time

of about 6 h, the mixture was heated at 70 °C to 80 °C for about 30 sec. The samples were

centrifuged and re-dispersed in hexane three times. Three kinds of nanostructures were further

6

used to modify the electrode surfaces as described in sections 2.3.2 (citrate-stabilized NP) and

2.3.3 (OANP and OANW).

The electrode surfaces were structurally characterized by scanning electron

microscopy (SEM) (Gemini 1550 VP, Carl Zeiss, Jena, Germany) and transmission electron

microscopy (TEM) (FEI CM20 microscope operated at 200 kV, by drop casting particle

dispersions on copper grids coated with Formvar film).

2.3. Electrode preparation.

2.3.1. Flat thin-film gold electrodes. For the preparation of thin-film gold working

electrodes (WE), a silicon oxide layer 1 µm in thickness was grown on a silicon substrate.

Thin films of titanium as the adhesion layer (10 nm) and gold (300 nm) were prepared on

Si/SiO2 substrates by sputter deposition. The electrodes were cleaned in acetone, propanol,

water, and H2O2:H2SO4 1:2 v/v and rinsed thoroughly with water. After chemical cleaning,

the substrates were subjected to electrochemical cleaning by consecutive potential cycles in

0.1 M H2SO4 between 0 V and 1.5 V at 0.05 V s-1 starting and ending at 0 V against a

Ag/AgCl/KCl 3 M reference electrode. These electrodes are referred to as thin-film flat

electrodes (without the immobilized nanostructures). Subsequently, the thin-film electrodes

were used to prepare the electrodes with nanostructured surfaces.

2.3.2. Chemisorption of thiols and immobilization of citrate-stabilized AuNP. The

electrodes were immersed in 1 mM solutions of a thiol (ethanediathiol, benzenedithiol, or

cysteamine) in ethanol for 1 h. Thiols were chemisorbed at gold surfaces: Aun + RSH Aun-

1Au+S-. The electrodes were rinsed with ethanol and distilled water to eliminate excess thiols.

The thiol-modified electrodes were submersed in the solution of the citrate-stabilized gold

nanoparticles overnight and washed with water.

2.3.3. Immobilization of OA-stabilized gold nanostructures (NS). The OANP or

OANW samples prepared as described in section 2.2 were dropped onto the flat thin-film

electrode surface (prepared as described in 2.3.1) and left overnight to allow the

nanostructures to adhere. Subsequently, these electrodes were washed with hexane to remove

any nanostructures that had not adhered to the electrode surface. Oxygen plasma treatment

was performed in a plasma oven (diener electronic), 200 watt, 0.7 mbar to remove a

stabilizing agent from the surface of the nanostructures.

2.4. Immobilization of a metalloprotein cyt c. Thin-film flat and NS-modified

electrodes were immersed in solutions of 6-MHA/2-ME (5mM/5mM) in hexane for 1 h. The

electrodes were rinsed with hexane and phosphate buffer to eliminte excess alkanethiols. Cyt

c was electrostatically adsorbed from 30 µM solutions in phosphate buffer (4.4 mM, pH 7.0,

7

ionic strength 10 mM) at 4 ºC for 2 h. After adsorption, the substrates were rinsed with

phosphate buffer to remove any excess cyt c from the electrode surfaces.

2.5. Enzyme immobilization. The electrodes were immersed in 5 mM solutions of

cysteamine in hexane for 1 h. The electrodes were rinsed with hexane and phosphate buffer to

eliminate excess alkanethiols. The electrodes were further immersed in 2.5 % v/v

glutaraldehyde solution in phosphate buffer (0.1 M, pH 7.0) for 1.5 h at room temperature.

The electrodes were rinsed with phosphate buffer and immersed in the solution of HRP (5

mg/ml) for 1 h at room temperature and left at 4 ºC overnight. After immobilization, the

substrates were rinsed with phosphate buffer to remove any excess enzyme from the electrode

surface.

2.6. Electrochemical measurements. Cyclic voltammetry measurements were

performed in a three-electrode setup controlled by a potentiostat (AUTOLAB, Eco Chemie,

The Netherlands). The electrochemical cell was composed of a gold working electrode, coiled

platinum wire counter electrode, and a Ag/AgCl reference electrode (3 M KCl, Ef = 0.210 V

against NHE). Electrochemical experiments were performed at room temperature 21±1 ºC.

Solutions were deaerated with argon and maintained under an argon stream during the

measurements. The diameter of the working electrode in the electrochemical cell was 0.5 cm.

2.7. Correction of peak potential. The anodic and cathodic peak potentials of cyt c

on nanostructured electrodes were corrected for the uncompensated ohmic potential drop due

the solution resistance according to the equation: Epcor = Ep

CV - Ip.Ru, where Ep

CV is the Ep

value defined from the voltammogram and Ru is the uncompensated resistance [51]. Peak

potentials corrected using this equation are shown in Table 1.

2.8. Surface coverage of cyt c. For a redox couple that is immobilized on the

electrode surface, the surface coverage on the electrode surface, o, is given by:

nFA

0

,

where o is the surface coverage of cyt c on the electrode surface, Α is the area under the peak

(in units V.A), n is the number of electron transfer, A is the area of the flat electrode in the

electrochemical cell (0.196 cm2), and ν is the scan rate. The surface coverage was calculated

from the baseline-corrected peaks.

3. Results and discussion

8

3.1. Characterization of the Au NSs. The gold nanostructures on electrode surfaces

were analyzed by scanning and transmission electron microscopy (Fig. 1). The oleylamine-

stabilized gold nanoparticles were 10 to 14 nm in diameter, and citrate-stabilized gold

nanoparticles were 15 to 19 nm in diameter. The nanowires were about 2 nm in diameter and

up to several micrometers in length.

Figure 1. Scanning (A,C,E,F) and transmission (B,D) electron micrographs of (A, B)

Au NPs, (C,D) Au NWs, (E) and (F) citrate-stabilized NPs attached via cysteamine and

ethanedithiol, respectively. Scale bars are 100 nm (A,C), 5 nm (D), 20 nm (E), and 200 nm

(E,F).

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The electroactive surface area of the nanostructured electrode surfaces was determined

from the gold oxide reduction peak using electrochemical redox cycling, Figure 2 [3,52,53].

The electroactive surface area of the electrodes modified with nanostructures increased about

5.8 (S = 1.15 9% cm2), 6.2 (S = 1.22 14% cm2), and 3.5 (S = 0.712 12% cm2) times

compared to a planar thin film gold electrode surface (S = 0.196 0.014 cm2) for OANP,

OANW, and cit-NP electrodes, respectively.

Figure 2. Oxidation and reduction of electrode surfaces in 0.1 M H2SO4: planar gold thin film

(1), planar gold film modified with citrate-stabilized Au NPs covalently attached to the thin

film surface by EDT (2), planar gold thin film modified with OANPs (3) and OANWs (4),

scan rate - 30 mV.s-1. OP treatment 5 min.

3.2. Direct electrochemistry of cyt c.

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Scheme 1. Assembly of metalloproteins on electrodes: (A) cyt c, and (B)

oxidoreductase enzyme horseradish peroxidase (structures of horse heart cyt c and horseradish

peroxidase were adapted from the Protein Data Bank [54]).

The –COOH and –NH2-terminated monolayers of thiols on gold (Sections 2.4 and 2.5)

were used for the immobilization of proteins.

Cyt c was immobilized on the electrode surfaces as described in Section 2.4, Scheme

1A [55]. During thiol chemisorption the stabilizing agent on the surface of the gold

nanostructures (molecules of oleylamine and its oligomeric reaction products or citrate ions)

were replaced by thiols with negatively charged carboxyl and alcohol groups. The

electrochemical characteristics of the heterogeneous electron transfer of cyt c and

concentrations of electroactive cyt c on the electrodes are compared in Figure 3 and Table 1.

Dependence of the redox currents of cyt c on the scan rate (Figure S1) proves that

electroactive cyt c was confined to the electrode surface.

Figure 3. CVs of cyt c on electrodes: planar thin-film Au-SAM-cyt c electrode. A:

planar thin-film Au-SAM-cyt c (a), Au-NPs-OP-SAM-cyt c (b), planar thin-film Au-NWs-

OP-SAM-cyt c (c), planar thin-film Au-SAM without cyt c (d, dotted line). B: planar thin-film

Au-SAM-cyt c (a), Cys-bound cit-Au-NPs-SAM-cyt c (b), EDT bound cit-Au-NPs-SAM-cyt

c (c); other conditions: scan rate - 50 mV s-1, 4.4 mM phosphate buffer, pH 7.0.

The slight decrease in the electron transfer rates for the electrodes modified with

nanostructures is probably due to an additional tunneling barrier for the electron transfer

provided by organic stabilizing molecules remaining on the surfaces of nanostructures. This is

more pronounced for the OA-stabilized nanostructures. The amount of cyt c immobilized on

the AuNS electrodes was significantly higher than for the thin-film flat electrodes, Table 1,

with the highest amount of cyt c immobilized on the OA-stabilized nanoparticles. For the

11

citrate-stabilized nanoparticles, faster kinetics of the electron transfer and larger amount of cyt

c was observed for the nanoparticles electrostatically immobilized on the thin-film gold

electrode surface using cysteamine. Electrodes with citrate NPs covalently bound to the

electrode surface using EDT demonstrated worse characteristics, Table 1. Electrodes modified

with Au-NSs display higher capacitive currents compared to the planar gold thin-film

electrodes (Figure 3) due to a greater surface area of the electrodes with immobilized gold

nanostructures (see Section 3.1).

Table 1. Electrochemical characteristics of the heterogeneous electron transfer of cyt c

on electrodes, oNS / o

flat – ratio of concentrations of cyt c on the OANS electrode and flat

electrode.

Electrode system Ecor, mV kETL, s-1 o, pmol cm-

2

oNS/o

flat

Au-SAM-cyt c 8(1) 9(1.1) 5.3(0.3) 1

Au-NP-OP-SAM-cyt c 39(5) 2.0(0.3) 94(8) 18

Au-NW-OP-SAM-cyt c 28(6) 2.4(0.5) 31(6) 6

EDT-cit-Au-NPs_SAM-cyt c 18(2.5) 3.2(0.4) 2.6(0.2) 0.5

Cys-cit-Au-NPs_SAM-cyt c 11(2) 5(0.6) 7.4(0.5) 1.4

a Ecor – difference between peak potentials corrected for the ohmic potential drop due to the

solution resistance; kETL – heterogeneous electron transfer constant found by the Laviron

method [56]; o – concentration of cyt c on the surface [53]; 95 % confidence intervals are

given in parenthesis (n=5).

For basic and applied studies, cyt c was immobilized on various flat and

nanostructured surfaces, e.g., gold nanostructures (mostly gold nanoparticles) [57-60],

graphene nanosheets [61], and fullerene film-modified electrodes [62]. However, the data on

the thermodynamics and kinetics of interfacial redox processes of cyt c electrostatically

adsorbed on the surfaces revealed a large scattering of the data for the formal redox potential

E0’, the heterogeneous electron transfer rate constant kET, and the amount of the immobilized

electroactive cyt c. The heterogeneous electron transfer rate constant of cyt c electrostatically

adsorbed on thiol SAMs varied in the range of 0.3 s-1 – 880 s-1 [59,60]. The heterogeneous

12

electron transfer rate constant decreased with an increasing electron transfer distance

(alkanethiol chain length) [63,64]. Additionally, some discrepancy in the kET values may be

explained by the observed dependence of kET on v (scan rate) [65]. High surface coverage of

cyt c up to about 123 pmol cm2 in the layered nanospace of graphene nanosheets [61]

compared to the theoretical value of 13 pmol cm-2 for monolayer cyt c on the GCE surface

and 20 pmol cm-2 [66] on a gold surface was reported. Lower values are usually reported for

the electroactive coverage on SAM-modified flat gold surface.

3.3. Hydrogen peroxide sensing with horseradish peroxidase (HRP) enzyme.

Covalent attachment of the enzymes to the electrode surface improves the stability and

reproducibility of the enzyme-modified electrodes in comparison to the adsorption, therefore

this strategy was employed here. The HRP enzyme was immobilized on the flat thin-film and

NS electrodes, as described in Section 2.5 and Scheme 1B [67]. The electrocatalytic

properties of the sensors were evaluated based on the determination of hydrogen peroxide in

the presence of the redox mediator hydroquinone, since no stable and reproducible direct

electron transfer between the catalytic center of the enzyme and electrodes was observed in

the present work. It was shown earlier that the cationic peroxidases, such as HRP,

demonstrate a lower percentage of molecules in direct electron transfer on graphite electrodes

than the anionic peroxidases [42]. The redox mediator hydroquinone participates in the

reaction as a two electron-proton mediator and demonstrates a higher reaction rate between

the oxidized HRP and hydroquinone (k= 1.2.107 M-1 s-1 [68,69]) in comparison with other

mediators.

For the HRP-modified electrodes, the dependence of the reduction current of the

mediator (Q + 2e + 2H+ QH2) on the concentration of hydrogen peroxide is described in

Scheme 1B. The dependence of the redox currents of the sensor response on the scan rate is

shown in Figure S2.

Figure 5 shows the calibration curves for the enzyme biosensors based on flat thin-

film and nanostructure-modified electrodes. Sensors based on HRP immobilized on the

electrodes with nanostructures display significantly improved performance with respect to the

sensitivity and working concentration range (Figure 5, Table 2). A working concentration

range of 18 to 500 µM and detection limits of 5 µM (RSD 0.09) for the HRP-OANW, 8 µM

(RSD 0.09) for the HRP-OANP, and 14 µM (0.15) for the HRP-cit-NP sensors are

13

comparable with the characteristics of the hydrogen peroxide nanostructured electrodes

reported recently [44,70-76]. Higher current densities were observed for the electrodes

modified with nanostructures compared to the HRP-modified flat thin-film gold electrode.

This can be explained by the larger amount of electroactive enzyme HRP on the

nanostructured electrodes.

Figure 4. Dependence of the current on the concentration of hydrogen peroxide for

OANP electrodes. Concentrations of hydrogen peroxides: (a) 0 M, (b) 20 µM, (c) 50 µM, (d)

125 µM. Other conditions: 2.5 % GA, deaerated solutions, scan rate 50 mV s-1, phosphate

buffer 0.1 M, pH 6.8, 1.0 mM QH2.

Figure 5. Calibration curves of HRP sensors (a) HRP-Au, (b) HRP-OANP, (c) HRP-

OANW, and (d) HRP-cit-NP. Other conditions: 2.5 % GA, deaerated solutions, scan rate 50

mV s-1, phosphate buffer 0.1 M, pH 6.8, 1.0 mM QH2. Error bars represent the confidence

limits (p=0.95, n=5) at each concentration point.

Table 2. Detection limit, sensitivity, and long-term stability of the HRP biosensors.1

HRP sensor Sensitivity,

A M-1 cm-2

Detection limit,

µM 2,3

Long-term stability,

% 3

14

HRP-Au thin-film gold 0.012(0.005) 16 (0.12) 82

HRP-OANP 0.027(0.005) 8 (0.09) 91

HRP-OANW 0.031(0.004) 5 (0.09) 91

HRP-cit-NP 0.22(0.0035) 14 (0.12) 90

1 - Relative standard deviations are given in parenthesis. 2 - S/N=3, relative standard

deviations of the determinations near the detection limit were 9 to 12 %. 3 - % of the sensor

response to 50 µM H2O2 after a storage period of one month.

The dependence of the sensor response on the pH of the solution was studied in the pH

range from 4 to 8, with a maximum current response being observed in a pH interval of 6 to 7

for all sensors. The reproducibility of the sensors was evaluated by measuring sensor response

in 125 µM H2O2. The relative standard deviation of five successive measurements was 3 %.

The HRP thin-film, HRP-OANP, HRP-OANW, and HRP-cit-NP sensors retained about 82,

91, 91, and 90 % of their biocatalytic response, respectively, during storage in a phosphate

buffer at 4 °C (Table 2).

The sensors for nanomolar concentrations of hydrogen peroxide have been also

reported based on various materials and materials combinations [45,46], including conducting

charge-transfer complex TTF-TCQN [77] or Prussian blue [78,79]. We expect that studies on

the surface chemistry and assembly of OA nanostructures as well as protein immobilization

may further enhance the electrochemical and biosensing properties of cyt c and HRP

electrodes.

Hydrogen peroxide enzyme biosensors based on the electrodes modified with

nanostructures demonstrate improved performance in comparison to the flat thin-film

electrodes due to: 1) geometrical signal enhancement, i.e. greater effective surface area, and

as a consequence, a larger amount of immobilized proteins on nanostructured surfaces, and 2)

nanostructured surfaces provide a favorable microenvironment for preserving electrochemical

and biocatalytic activity of metalloproteins.

Conclusions

This paper compares nanostructure platforms based on the charge(citrate)-stabilized

and sterically(OA)-stabilized gold nanostructures for biosensing and bioelectrochemical

systems. Heme-containing metalloproteins cyt c and oxidoreductase enzyme HRP on

nanostructured interfaces were immobilized on electrodes and their electrochemical properties

were studied. The nanostructured electrodes provided a good environment for stable and

15

reproducible immobilization of electroactive metalloproteins. Enhancement of the

electrochemical signal was achieved due to a greater electroactive surface area of the

nanostructures. The heterogeneous direct electron transfer of cytochrome c on electrodes was

studied and compared for two types of gold nanostructures. Higher sensitivity,

reproducibility, and long-term stability were observed for the detection of H2O2 with HRP

sensors in the case of sensors with electrode surfaces modified with nanostructures compared

to a flat thin-film HRP sensor.

Acknowledgements. We would like to thank S. Lenk, R. Voschinsky, M. Banzet, and

E. Brauweiler-Reuters for TEM and SEM imaging, citrate-stabilized gold nanoparticles, and

preparation of the thin-film gold electrodes. Financial support of the RFBR 14-03-01079

(Russian Foundation for Basic Research) is gratefully acknowledged.

Supporting information: Dependence of Ip on the scan rate for the heterogeneous

direct electron transfer of cyt c on the electrodes (Figure S1), Dependence of the HRP-OANP

sensor response on the scan rate in the presence of 200 µM H2O2 (Figure S2).

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