A amperometric biosensor for hydrogen peroxide by adsorption of horseradish peroxidase onto single-walled carbon nanotubes
Graphical abstract
Highlights
► A low detection limit and novel nanostructured electrochemical biosensor based on the integrated assembly of HRP and SWNTs is described. ► SWNTs dispersed with sodium cholate adsorbed HRP using noncovalent method by dialysing suspension and the benefit of noncovalent immobilization is what allows for the electronic structures and the geometric structures of the SWNTs to be retained. ► Direct electrical contacting of redox proteins to electrodes has been accomplished through self-assembling layers and noncovalent conjugates which provide electron-conducting pathways between the protein and the electrode.
Introduction
Electrical communication between redox sites of enzyme and electrode is the basis for developing various amperometric biosensor devices [1]. Nevertheless, direct electron transfer is usually impossible with most enzymes because their redox centers are located deep inside the insulated protein shells. Therefore, some carbon materials, such as carbon nanotubes [2], [3], carbon nanofibers [4], and highly ordered mesoporous carbons [5], [6], are required in order to establish an electrical connection between these redox centers and the electrode [7] for fabricating a third-generation enzyme biosensor.
Single-walled carbon nanotubes (SWNTs) possess highly unique electronic, mechanical, and optical properties, making them the subject of intense study because of their discovery in 1993 by Iijima [8], [9]. A large length-to-diameter ratio and a good conductivity of the SWNTs, make it possible to form a three-dimensional conducting matrix that can be used for immobilization of enzymes and proteins, more importantly, direct electron transfer of those biomacromolecules [10] enhanced faradaic responses [11]. However, As-synthesized single-walled carbon nanotubes (SWNTs) are bundled, preventing their efficient application [12]. To improve upon the properties of the SWNTs, low-cost and simple approaches utilizing a sodium cholate suspension-dialysis process to their debundled modification have been much pursued vigorously in recent times [13], [14], [15]. In this way, the proteins were adsorbed to form a stable solution-phase complex with SWNTs, neither the native structure and biological activity of proteins nor the unique propertie of SWNTs was lost.
The functionalized single-walled carbon nanotubes (SWNTs) are promising materials as carriers for enzyme immobilization [16], [17]. The main advantage of using functionalized SWNTs is that the biomolecules can be immobilized firmly onto the SWNTs through covalent binding at the functional sites or noncovalent methods. Benefits of covalent immobilization are that it provides a strong linkage of the protein to the SWNTs and may increase the stability of the protein [18]. However, covalent coupling normally damages the sidewalls of SWNTs and disrupts their unique, electrical and optical properties [19], [20], [21]. In contrast, noncovalent methods can allow for the electronic structures and the geometric structures of the SWNTs [14], [22], [23], [24], [25], [26] to be retained. Recently, a noncovalent method of protein attachment to SWNTs utilizing a sodium cholate suspension-dialysis process has been developed [13], [14]. This method has been successful in coupling both the enzyme glucose oxidase [13] and horseradish peroxidase to SWNTs [14].
Among many different strategies used for the modification of electrode surfaces, the self-assembly technique exhibits unprecedented flexibility to tailor interfacial properties and presents biologically relevant groups, which makes it well-suited for the control of biomolecular density and orientation at the solid-liquid interface to obtain better reproducibility and higher efficiency [25]. It is well known that alkyl or aromatic disulfides and thiols form a close-packed ordered monolayer, “self-assembled monolayer” (SAM), on gold or silver surfaces via chemisorptive S–Au or S–Ag bonds [27], [28], [29]. The strength of this covalent bond is 40 kcal/mol [30]. Based on this principle, l-cysteine can form self-assembled monolayers on gold surfaces.
The direct electron transfer of immobilized proteins such as HRP [31], cytochrome C [32], and hemoglobin [33] with regard to Fe(III) to Fe(II) conversion has been well studied and extensively employed for biosensing and the preparation of mediator-free biosensors [34]. Direct electrical contacting of redox proteins to electrodes has been accomplished through self-assembling monolayers, polycation layering, sol–gel and conducting polymers, clay colloids, or carbon paste, all of which provide electron-conducting pathways between the protein and the electrode [35], [36], [37], [38], [39], [40], [41], [42]. The relatively low amperometric detection limit obtained for l-cysteine–HRP–SWNTs-modified electrode is lower than the 2.5 × 10−7 M reported for HRP immobilized in ZrO2-Grafted Collagen/DMSO Membranes [43], 1 × 10−7 M for HRP–CPE incorporating the graphite powder [44], and 2.5 × 10−7 M for HRP-Chi-BMIM·BF4/GC electrode [45]. Recently, a method to determine hydrogen peroxide in macrophage RAW 264.7 cell extracts with the detection limit of 5.6 nM were reported [46].
Hydrogen peroxide which present in aerobic cells as a metabolite in low concentrations is generated by nonenzymatic and superoxide dismutase-catalyzed dismutation reactions. The active oxygen species H2O2, superoxide anion and hydroxyl radical have been implicated as a cause of cellular death [47]. H2O2 causes DNA single-strand break in human fibroblasts. Thus, the method to determine H2O2 of low concentration is desired for constructing. In the present study, by combining the advantageous features of SWNTs and S–Au, a novel H2O2 biosensor has been constructed. The advantages of l-cysteine–HRP–SWNTs-modified gold electrode are the low detection limit and the high activity of HRP. We have developed a hydrogen peroxide biosensor, in which horseradish peroxidase was immobilized onto a functionalized SWNT through noncovalent method and the carboxylic groups of the functionalized SWNT was immobilized onto an amino-terminated l-cysteine self-assembled monolayer (SAM) performed on the Au electrode. The low detection limit obtained in this work is attributed to the intimate contact resulting from the close proximity between enzyme and electrode, attached by SWNTs.
Section snippets
Materials
Single-walled carbon nanotubes were purchased from Nanoport. Co. Ltd. (Shenzhen, China). Horseradish peroxidase (HRP) was obtained from Sigma. l-Cysteine, sodium cholate, N-(3-dimethylamino)propyl-N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) were purchased from Aldrich. A phosphate buffer saline solution (PBS) was prepared by modifyng 0.01 M disodium hydrogen phosphate (Aldrich) with the admixture of 0.01 M sodium dihydrogen phosphate (Aldrich) with 0.1 M sodium
Characterization of the l-cysteine–HRP–SWNTs-modified gold film
SEM is a powerful tool to characterize the morphologies of different composites. Fig. 2 shows the SEM images of the l-cysteine-modified gold film before (Fig. 2a) and after integrating the HRP–SWNTs (Fig. 2b). After the covalence of the HRP–SWNTs (Fig. 2b), SWNTs were seen to be distributed on the surface of l-cysteine-modified gold film and some of them intercalated into the interior film.
Electrochemical characteristics of the modified electrode
Cyclic voltammetry (CV) is especially well-suited to study of electron-transfer kinetics of electroactive
Conclusion
In this study, we demonstrate that HRP–SWNTs conjugates produced by the sodium cholate suspension-dialysis method were highly dispersed and retained a significant amount of native enzymatic activity. We also demonstrate that these HRP–SWNT conjugates can be combined with l-cysteine assembled on the gold electrode by the self-assembly technique to fabricate highly sensitive sensors to H2O2. HRP–SWNTs were constructed in order to improve the enzyme immobilization on the substrate. A most
Acknowledgments
This work was supported by the Natural Science Foundation of China (Nos. 21175108, 20875077 and 20927004), the Natural Science Foundation of Gansu (No. 0701RJZA109) and Key Laboratory of Polymer Materials of Gansu Province, China.
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