Elsevier

Talanta

Volume 78, Issue 1, 15 April 2009, Pages 248-252
Talanta

Direct electrochemistry of cytochrome c on a phosphonic acid terminated self-assembled monolayers

https://doi.org/10.1016/j.talanta.2008.11.006Get rights and content

Abstract

The direct electrochemistry of cytochrome c (cyt c) on a gold electrode modified with 3-mercaptopropylphosphonic acid [HS–(CH2)3–PO3H2, MPPA] self-assembled monolayers (SAMs) was for the first time investigated. Electrochemical measurements and surface-enhanced infrared absorption spectroscopic reveal that the adsorption kinetics of cyt c on the MPPA-SAMs is very fast (saturation adsorption is completed within 5 s) and the immobilized cyt c molecules retain their native secondary protein structure. The nature of interaction between cyt c and –PO3H2 groups is mainly the electrostatic interaction. The direct electrochemistry of the immobilized cyt c on the –PO3H2 terminated SAMs with short chain is nearly reversible. Its formal potential (E0 = 18 ± 3 mV vs. SCE) is very close to that of cyt c in an aqueous solution (E0 = 18–22 mV vs. SCE). In addition, the electron transfer rate of cyt c immobilized on –PO3H2 terminated SAMs is relatively slow as compared to –SO3H and –COOH terminated SAMs, indicating excess negative charge density on the SAMs surface will decrease the electron transfer rate of cyt c.

Introduction

Among the electroactive proteins, cytochrome c (cyt c) is usually used as a model system to study the direct electron transfer. Particularly, the direct electrochemistry of cyt c immobilized on metals coated with monolayers of organic molecules has attracted extensive attentions of several groups over the past few years, because these simple systems allow for a systematic study of the parameters that control the electron transfer kinetics. Self-assembled monolayers (SAMs) with –COOH [1] and –SO3H [2] terminated thiolates, which provide a negatively charged surface for cyt c adsorption, have been extensively study to understand the electron transfer properties of electrostatically complexed cyt c and draw analogies to its physiological redox partners. As another very important anion (i.e. –PO3H2) terminated SAMs, there is no formal report on the electron transfer of cyt c although Murgida and his co-workers repetitiously proposed that electron transfer of cyt c on a Ag electrode modified with –PO3H2 terminated thiolates with long carbon chains can hardly occur [3], [4].

Phosphate is an essential component in biologic system due to its pervasive existence in organs and tissues, such as cell membranes, bones and body fluids. For example, a lipid bilayer membrane is composed of phospholipids containing various phosphate head groups. Based on the biomimetic concepts, the phosphate and phosphonate terminated SAMs have been studied. Tanahashi and Matsuda [5] reported that the negative charged functionalities strongly induced apatite formation on a –PO3H2 interface. Lin and co-workers [6] found that the ionic –PO3H2 surface exhibited a lower platelet reactivity than the –COOH. These studies show that the –PO3H2 groups play very important roles in biological systems.

In the present work, we for the first time study the immobilization of cyt c on a –PO3H2 terminated SAMs on an Au substrate. Electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to characterize the immobilization of –PO3H2 functional interface on the gold electrode. Surface-enhanced infrared absorption spectroscopy (SEIRAS) affirms that the cyt c immobilized on MPPA-SAMs retain their native secondary protein structure. Voltammetry was used to understand the interaction between cyt c and surface-immobilized –PO3H2 groups, and the direct electron transfer behavior of the cyt c molecules immobilized on the –PO3H2 terminated SAMs.

Section snippets

Materials

All the chemicals were of analytical grade (AR) and used without further purification. Cytrochrome c from a horse heart (type VI) was purchased from Sigma–Aldrich (St. Louis, USA). 11-Mercaptoundecanylphosphonic acid [HS–(CH2)11–PO3H2, MUPA] was synthesized according to the process reported in the literature [7]. 3-Mercaptopropylphosphonic acid [HS–(CH2)3–PO3H2, MPPA] was prepared according to the modified procedure by using 3-bromopropylene as the starting material [7].

Preparation of MPPA/Au electrode

After the Au electrode

XPS characterization of MPPA/Au electrode

XPS was used to monitor the characteristic elements of the assembled electrode surface and to confirm the successful formation of the S–Au bond. Fig. 1a presents the XPS spectra of the MPPA/Au in the S(2p) region. An asymmetric peak that can be fitted as a spin doublet at 162.0 and 163.2 eV is observed, which corresponds to the S2p3/2 and S2p1/2 peaks, in agreement with the reported values for the thiolates SAMs systems on a gold surface [6]. XPS scan of the P(2p) region shows a peak that is

Conclusion

The cyt c immobilized on the –PO3H2 terminated SAMs via the electrostatic interactions retains its native secondary protein structure and the direct electron transfer can occur. The direct electrochemistry of the immobilized cyt c on MPPA/Au is nearly reversible, and its formal potential (E0 = 18 ± 3 mV vs. SCE) is very close to that of cyt c in an aqueous solution (E0 = 18–22 mV vs. SCE). Compared with pervious reported works, the present experimental results demonstrate that the value of formal

Acknowledgments

This work is supported by NSFC (Nos. 20325103, 20775034, 20535010), the National Science Fund for Creative Research Groups (20521503, 20721002) and the National Basic Research Program of China (2007CB925102).

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