Bio-Medical Materials and Engineering 24 (2014) 1079–1084 DOI 10.3233/BME-130906 IOS Press

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Direct electron transfer of horseradish peroxidase on a functional nanocomplex modified glassy carbon electrode Bao-Lin Xiaoa, Jun Honga,*,Yun-Fei Gaoa, Tian Yanga, Ali Akbar Moosavi-Movahedib and Hedayatollah Ghourchianb a b

School of Life Sciences, Henan University, Kaifeng 475000, Henan, PR China Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran

Abstract.Direct electron transfer of horseradish peroxidase (HRP) was achieved by immobilizing HRP on a functional nanocomplex modified glassy carbon (GC) electrode. The cyclic voltammograms (CVs) of the modified electrode have a pair of well-defined redox peaks with a formal potential (E') of -26±2 mV versus Ag/AgCl, in 0.05M, pH7.0 phosphate buffer solution (PBS) at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was calculated to be 1.94 s1. The modified electrode response toward hydrogen peroxide was linear in the concentrations ranging from 0.28 M to 10 M, with a detection limit of 0.28 M. The apparent Michaelis–Menten constant (Kmapp) for H2O2 was 2.54 M. Moreover, results of biochemical computation showed that the amino acid residues (Ala34, Arg38, Ser73, Arg75, Ala140, Pro141, Phe172, Gly173, Lys174, Phe179, Arg31, Ser35, Lys174, Gln176) of HRP may playa crucial role in the improvement of electron transport between electro-active site (heme group) of an HRP molecule and nanocomplex modified GC electrode. Keywords: Horseradish peroxidase, direct electron transfer, gold nanoparticles, carboxylic multi-walled carbon nanotubes; biochemical computation

1. Introduction Horseradish peroxidase (HRP) is an important heme containing enzyme that has been widely used in different hydrogen peroxide biosensors [1-4]. Carbon nanotubes (CNTs) and gold nano particles (AuNPs) are widely used for the fabrication of electrochemical biosensors in recent years, due to their excellent electron transport properties, large ratio surface area and perfect biocompatibility [2-14]. In this study, direct electron transfer of HRP was achieved and studied when HRP was immobilized on a functional nanocomplex-modified glassy carbon (GC) electrode. This electrode could be used as a high sensitivity hydrogen peroxide (H2O2) biosensor.

*

Corresponding author. E-mail address: [email protected]

0959-2989/14/$27.50 © 2014 – IOS Press and the authors. All rights reserved

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B.-L. Xiao et al. / Direct electron transfer of HRP on a functional nanocomplex modified GC electrode

2. Experimental 2.1. Reagents and Apparatus HRP(RZ㸼3.0, 250U/mg), Nafion (NF, 5% ethanol solution), L-cysteine (Cys), HAuCl4, sodium citrate and hydrogen peroxide (v/v 30%) were obtained from Sigma, Saint. Louis, MO, USA. MWCNTs was purchased from Shenzhen Nanotech Port Co., Ltd, China. Other chemicals were of analytical grade and used without further purification. Double distilled water was used throughout the studies. Electrochemical measurements were carried out in a three electrode cell powered by an electrochemical system comprising of CHI650C (Shanghai CHI Instrument Co. Ltd., China). A GC electrode of 3 mm diameter, an Ag/AgCl and a platinum wire were served as the working, reference and counter electrodes, respectively. The electrochemical measurements were carried out in a N2-saturated 05 M sodium phosphate buffer solution (PBS, pH 7.0) at 25°C. 2.2. Fabrication of NF/HRP/CMWCNTs/Cys/AuNPs/GC electrode AuNPs were prepared as previously reported in the literature [15-18]. The mean size of AuNPs was evaluated to be 18.4 nm (data not shown). The MWCNTs were functionalized according to the reference [19]. The AuNPs were electro-deposited on the surface of a clean bare GC electrode (voltage range: 0~1.1 V, scan rate: 0.1V/s, 25 cycles). The GC electrode was then dipped in a freshly prepared 10 mM Cys solution for 20 min, washed with water carefully, 3 l of carboxylic acid functionalized multi-walled carbon nanotubes (CMWCNTs) (2 mg/ml) was dropped onto the surface of the electrode, and dried at 25ºC. Then, the modified electrode was dipped in a HRP solution (12 mg /ml) for 12 hours at 4 ºC. Finally, the electrode surface was covered by 2 l of NF (5% in ethanol). 2.3. Biochemical computation by using Ligplot [20] and Discovery Studio® (DS) Ligplot is a program for automatically plotting protein-ligand interactions. This program can generate schematic diagrams automatically of protein-ligand interactions from a given PDB file. The operating manual is located at the site: http://www.biochem.ucl.ac.uk/bsm/ligplot/manual/. DS, built on Accelrys’ Pipeline Pilot technology, is a software suite of life science molecular design solutions for computational chemists and computational biologists. (http://accelrys.com/) 3. Results and Discussion Fig. 1A shows the cyclic voltammograms (CVs) of: (a) NF/HRP/CMWCNTs/Cys/AuNPs/GC electrode; (b)NF/HRP/CMWCNTS/GC electrode and (c) bare GC electrodes. It can be seen that NF/HRP/CMWCNTs/Cys/AuNPs/GC electrode has a pair of well-defined redox waves with a formal potential [Eº'=(Epa+ Epc)/2] of -26±2mV (versus Ag/AgCl). This value is greater than the Eº' obtained for native HRP in solution (-270 mV versus NHE at pH7.0) [21] and HRP nano-particles at a gold electrode(70 mV versus Ag/AgCl at pH7.4) [22], but smaller than that Eº' at a gold colloid/Cys/NF modified platinum disk electrode (451 mV versus NHE) [23] and HRP/NF-Cys/Au electrode (60 mV versus Ag/AgCl) [24]. CVs of electrode (a) have a pair of stronger redox peaks compared with that of

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B.-L. Xiao et al. / Direct electron transfer of HRP on a functional nanocomplex modified GC electrode

electrode (b), and the AuNPs could help to increase the redox peak currents significantly. Moreover, the direct electron transfer of HRP at a bare GC electrode was too slow to be observed in (c). 50

50 30

150

10 Inne r

-10

-30

2

0.3

0

R = 0.9933

0

C

-150

-30

A

R = 0.9981

Ep / V

c -10

y = 0.1481x + 0.2578

2

Ip /A

I/A

b

10

0.6 y = 204.66x + 5.3972

30 I / A

300

O ute r

a

-0.3

y = -172.79x - 11.571

B

-50 0.4

0

-0.4

E (vs Ag/AgCl) /V

-0.8

2

R = 0.9957

-300

-50 0.8

0.8

0.4 0 -0.4 E (vs Ag/AgCl) / V

-0.6

0

-0.8

D

y = -0.1795x - 0.4402

2

R = 0.9973

0.5 1 -1 Scan rate  / V s

1.5

-3

-2

-1 ln ( )

0

1

Fig.1 (A) CVs of (a) NF/HRP/CMWCNTs/Cys/AuNPs/GC electrode; (b) NF/HRP/CMWCNTs/GC electrode; (c) bare GC electrode, respectively, in 0.05 M PBS (pH 7.0) at a scan rate of 0.05 V/s. (B) CVs of NF/HRP/CMWCNTs/Cys/AuNPs/GC electrode in 0.05M PBS (pH 7.0) at different scan rates(from inner to outer): 0.02,0.04,0.06 … 1.2 V/s, respectively. (C) Plot of peak current (Ip) versus scan rates (). (D) Plot of peak potential (Ep) versus logarithm of .

CVs of NF/HRP/CMWCNTs/Cys/AuNPs/GC electrode at various scan rates are shown in Fig. 1B. The peak currents increased linearly with the increasing scan rate () (Fig. 1C), indicating that HRP is confined on the nanocomplex modified GC electrode [24]. The cathodic peak potential in the range from 0.5 to 1.4 V/s changed linearly versus ln (Fig. 1D). According to Equation (1)[25]: E p = E °'+

RT RT − ln v anF anF

(1)

Where , n, T, R and F are cathodic electron transfer coefficient, number of electrons, temperature (293 K here), gas constant (8.314 JK1mol1) and Faraday constant (96,485 C½mol1), respectively. It could be calculated that n=1, a=0.329[26]. The apparent heterogeneous electron transfer rate constant (ks) was calculated to be 1.94 s-1 according to the Equation (2) [27]: ln k s = a ln(1 − a ) + (1 − a) ln a − ln(

nFΔΕ p RT ) − a (1 − a ) nFv RT

(2)

The average surface concentration () of electro-active sites (heme groups) of HRP on the surface of GC electrode could be estimated to be 4.97×10-10 mol/cm2 based on the slope of Ip versus  in Equation (3):

Ip =

n 2 F 2 AΓv 4 RT

(3)

The value is higher than the  value of monolayer of HRP (5.0×10–11 mol/cm–2)[28,29]. This high surface concentration can be attributed to the larger surface area and good biocompatibility of the nanocomplex. The cathodic peak current of the nanocomplex modified GC electrode was recorded in 0.05 M PBS (pH7) containing different concentration of H2O2 (0.03-10 M). The cathodic peak current increases with the increasing concentration of H2O2 (Fig. 2A). The detection limit was 0.28 M (Fig. 2B). The cathodic peak currents of the modified electrode were linearly proportional to the concentrations of H2O2 ranging from 0.28 to 10 M (Fig. 2C).

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B.-L. Xiao et al. / Direct electron transfer of HRP on a functional nanocomplex modified GC electrode

The apparent Michaelis–Menten constant (Kmapp ) was calculated to be 2.54 M (Fig. 2D) from the electrochemical version of Linewearver–Burk Equation (4)[24]:

1 1 K app = + m I s I max I max c

(4)

y = 1.7021x + 27.114 2

35

Ipc / A

R = 0.9991

Ipc/A

40

Detection Limit

28 B

A

30

1/I / A

45

29

y = 0.0333x + 0.0203

40

02 0.

Ipc / A

45

04

50

0.

30

50

-1

Where, c, Is and Imax are H2O2 concentration in the bulk solution, steady-state current after the addition of H2O2 and maximum current measured under saturated substrate conditions, respectively.

35

-1/Km

30

app

1/Imax

C

D 0

27

25 0

20

40 60 80 [H2O2 ] / M

100

25

0

0.2 0.4 0.6 0.8 [H2O2] / M

1

1.2

0

2

4

6

8

[H2 O2 ] / M

10

12

-0.6

-0.4

-0.2

0

1/[H2 O2 ] / M

0.2

0.4

-1

Fig. 2 (A) The amperometric response of NF/HRP/CMWCNTs/Cys/AuNPs/GC electrode toward H2O2 in the concentration range from 0.03 to 10M. (B) Determination of the H2O2 detection limit for the modified GC electrode. (C) Linear range from 0.28 to 5 M. (D) Lineweaver–Burk plot for Kmapp determination.

Fig.3 (A) Hydrogen bond and hydrophobic interactions around the heme structure of an HRP molecule (PDB ID: 1H58). The figure is generated by using the software of Ligplot. The dashed lines represent hydrogen bonds, the spiked amino acid residues the hydrophobic interactions. (B) The three-dimensional schematic illustration of heme structure of the HRP molecule. The figure is generated by using the software of DS.

The operational stability of the modified GC electrode was evaluated by cyclic voltammetric method. The cathodic peak current was reduced by less than 5% after 50 cycles at a scan rate of 0.05 V/s, while the peak potential remained unchanged. For the storage stability, almost no change was observed in the CVs after three weeks of storage in a bottle over the PBS solution at 4|C (Data not shown).

B.-L. Xiao et al. / Direct electron transfer of HRP on a functional nanocomplex modified GC electrode

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Biochemical computation software was applied to simulate the important amino acids located in electron transfer channel between electro-active center (heme structure) of an HRP molecule and nanocomplex modified GC electrode. Fig. 3A and 3B show the heme structure of an HRP molecule (PDB ID: 1H58), which are generated by using Ligplot [20] and DS, respectively. The amino acid residues, which are located in the channel, and have hydrophobic interactions (e.g. Ala34, Arg38, Ser73, Arg75, Ala140, Pro141, Phe172, Gly173, Lys174, Phe179) and hydrogen bonds (e.g. Arg31, Ser35, Lys174, Gln176) with the heme group of the HRP molecule, may play a pivotal role in direct electron transport between the HRP molecule and nanocomplex modified GC electrode. 4. Conclusions Direct electron transfer HRP was achieved and studied on a functional nanocomplex modified GC electrode. The CMWCNTs/Cys/AuNPs nanocomplex acts as a promoter and bridge between the electroactive center of an HRP molecule and GC electrode. The modified GC electrode could be used as a third generation biosensor for determination of H2O2 with good sensitivity and stability. 5. Acknowledgements Financial support of Henan University Science Foundation is gratefully acknowledged. References [1]

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