Electrochemical assay of superoxide based on biomimetic enzyme at highly conductive TiO2 nanoneedles: from principle to applications in living cells

Yongping Luo , Yang Tian * and Qi Rui
Department of Chemistry, Tongji University, Siping Road 1239, Shanghai, 200092, China. E-mail: yangtian@mail.tongji.edu.cn; Fax: 86 21 65982287; Tel: 86 21 65987075

Received (in Cambridge, UK) 2nd February 2009 , Accepted 16th March 2009

First published on 7th April 2009


Abstract

A novel strategy for extremely durable, reliable, and selective in situ real-time determination of superoxide anion (O2˙) based on direct electron transfer of a biomimetic superoxide dismutase (SOD), Mn3(PO4)2, at highly conductive TiO2 nanoneedles is reported for the first time, and is further exploited for the determination of O2˙ from living normal and cancer ovary cells directly adhered onto the modified surface, suggesting that O2˙ might be proposed as a biomarker of cancer.


Superoxide anion (O2˙) is the primary reactive oxygen species (ROS), which are a class of radical and non-radical oxygen-containing molecules that display high reactivity with lipids, proteins and nucleic acids. Depending on concentration, location and context, ROS can be either “friends” or “foes”. Excessive ROS generation leads to apoptotic and necrotic cell death and pathogenesis of a panel of clinically distinct disorders including neurodegeneration, atherosclerosis, diabetes and cancer.1–2 Recently, a flurry of superoxide flash activity has been observed in different cell types after hypoxia, and it has been proposed that O2˙ could serve as a valuable biomarker for a wide variety of oxidative stress-related diseases.3 Therefore, quantitative analysis of O2˙in situ in real time is very important, not only for investigations to reveal the relevance of ROS to diseases, but also for routine health care, prevention of diseases, and selection of the appropriate remedy.

Up to date, a number of techniques for assaying O2˙ have been demonstrated, including ESR spin trapping, spectrophotometry, chemiluminescence and electrochemical methods.4–7 Electrochemical approaches have been gained more and more attention because of the striking advantages of simplicity, selectivity, low instrumental cost, and capability in real-time, and even in vivo detection. There have been many papers concerned with the electrochemical measurement of O2˙, typically based on the direct oxidation of O2˙, the oxidation of O2˙ mediated by enzymes such as cytochrome c, hemin , and superoxide dismutase (SOD).8–11 SOD-based third-generation biosensors developed on direct electron transfer of SODs have paved an elegant way for determination of O2˙ due to high specificity and selectivity.12–17 However, these kinds of biosensors based on enzymes are considered to be denatured or metabolized in real sample detection, although they have high selectivity and good sensitivity.

Herein we report a facile, but effective strategy for in situ analysis of O2˙ from ovary cells with high selectivity and relatively long stability, through direct electron transfer of a biomimetic SOD, manganese(II) phosphate (Mn3(PO4)2)18 realized at highly conductive TiO2 nanoneedles. The concept of this novel approach to determination of O2˙ is illustrated in Scheme 1(A). During the reaction of O2˙ by Mn3(PO4)2, two O2˙ ions are converted to one O2 molecule and one H2O2 molecule. Namely, one O2˙ oxidizes the Mn2+ to generate MnO2+ and H2O2, while another O2˙ reduces the MnO2+ to produce Mn2+ and O2. We can consider these two redox processes separately by fabricating two electrodes on which each reaction occurs separately. In the anodic process (Scheme 1(B)), the redox reaction between O2˙ and Mn2+ takes place to produce H2O2 and MnO2+. The generated MnO2+ can be reduced at the electrode. On the other hand, in the cathodic process (Scheme 1(C)), O2˙ reduces MnO2+ to produce Mn2+, which can be reoxidized at the electrode. Thus, by measuring the oxidation or reduction current at the Mn2+-modified electrode in the presence of O2˙, we can detect O2˙, that is, the present Mn2+-modified electrode, which is based on the specificity and selectivity of Mn3(PO4)2 for O2˙ as well as its direct electrode reaction, can be defined as a sensitive and selective biosensor for O2˙.


(A) Schematic illustration of O2˙− dismutation catalyzed by Mn2+/MnO2+. (B) The reduction of O2˙− to H2O2 and (C) the oxidation of O2˙− to O2 catalyzed by the Mn2+/MnO2+ redox couple confined into the Nafion-modified TiO2 nanoneedle electrode surface.
Scheme 1 (A) Schematic illustration of O2˙ dismutation catalyzed by Mn2+/MnO2+. (B) The reduction of O2˙ to H2O2 and (C) the oxidation of O2˙ to O2 catalyzed by the Mn2+/MnO2+ redox couple confined into the Nafion-modified TiO2 nanoneedle electrode surface.


          Cyclic voltammograms (CVs) obtained at (a) TiO2 nanoneedle film, (b) Nafion/TiO2 film, and (c, d) Mn2+/Nafion/TiO2 film in 0.1 M K3PO4 (pH 7.0) in the absence (a, b, c) and presence (d) of 150 μM O2˙−. Potential scan rate: 100 mV s−1.
Fig. 1 Cyclic voltammograms (CVs) obtained at (a) TiO2 nanoneedle film, (b) Nafion/TiO2 film, and (c, d) Mn2+/Nafion/TiO2 film in 0.1 M K3PO4 (pH 7.0) in the absence (a, b, c) and presence (d) of 150 μM O2˙. Potential scan rate: 100 mV s−1.

The E0′ values of the O2/O2˙ and O2˙/H2O2 redox couples are −0.35 and 0.68 V vs. Ag|AgCl and thus the E0′ of Mn3(PO4)2 should be located between these two values for mediating both the oxidation of O2˙ to O2 and the reduction of O2˙ to H2O2. For this purpose, several electrode matrices including glassy carbon, gold, TiO2 nanoneedles, and TiO2 nanoparticles were investigated (see ESI ). As a result, the TiO2 nanoneedle surface was selected as an electrode matrix for Mn2+ due to the appropriate E0′ of 552.4 ± 2.6 mV vs. Ag|AgCl and the high heterogenous rate constant of electron transfer. The TiO2 nanoneedle surface was characterized by SEM as demonstrated in Scheme 1(B, right part) and (C, left part). One couple of well-defined redox peaks was observed at the as-prepared Mn2+/Nafion/TiO2 surface in K3PO4 solution in the absence of O2˙ (Fig. 1(a)) while no electrochemical response was obtained at a bare TiO2 electrode (Fig. 1(b)) or at a Nafion/TiO2 film (Fig. 1(c)), indicating that the observed redox response is attributed to the Mn2+ adsorbed into the Nafion/TiO2 nanoneedle film. Meanwhile, both anodic and cathodic peak currents increase with the increasing potential scan rate, and are proportional to the scan rate (see ESI ), as expected for an electrochemical process with a surface-confined species. Moreover, the redox response has no intrinsic change during a continuous scan, e.g., at 100 mV s−1 for 2 h. These results demonstrate that Mn2+ is stably confined in the Nafion-modified TiO2 nanoneedles, and subsequently provide a durable platform for determination of O2˙. In the presence of O2˙, both anodic and cathodic peak currents corresponding to the redox reaction of Mn2+ clearly increased as shown in Fig. 1(d). The obvious increases in the anodic and cathodic peak currents could be ascribed to the oxidation and reduction of O2˙, respectively, as proposed in Scheme 1(B) and (C).

Both the anodic and cathodic current increase with the addition of O2˙ at the Mn2+/Nafion/TiO2 surface recorded by amperometry could be exploited as an electrochemical assay for O2˙. The typical steady-state currents at 0.6 V (oxidation) and 0.0 V (reduction) are proportional to the concentration of O2˙ in the range of 5 × 10−7–1.5 × 10−3 M, as depicted in Fig. 2(A) and (B), and the detection limit reached down to 170 nM. The wide linear detection range meets the requirement of O2˙ detection from normal physiological conditions (∼10−7 M) to morbid status (10−4–10−3 M); additionally, the response time is within 5 s. The interference of a variety of biorelevant analytes including Na+, Ca2+, K+, Mg2+, Zn2+, Fe3+, dopamine (DA), ascorbic acid (AA), uric acid (UA), H2O2 and cysteine (Cys), as well as O2 which exists extensively in biological systems, on determination of O2˙ was also investigated for control experiments. It was found that the degree of the interference on the determination of O2˙ is strongly dependent on the applied potential. At 0.6 V, 13.64, 24.81 and 4.62% anodic current from AA, DA and Cys was observed, however no obvious interference was observed with O2˙ detection at 0.0 V (see ESI ). Thus O2˙ could be cathodically detected at a Mn2+/Nafion/TiO2 film with high selectivity. Electrochemical control experiments were also carried out. No amperometric responses were observed at the bare TiO2 nanoneedles and Nafion-modified TiO2 nanoneedles with the addition of O2˙, indicating that neither TiO2 nanoneedles nor Nafion show a contribution to the amperometric current of O2˙. For the stability test, the response current for O2˙ was recorded three times daily and the current responses were found to be constant for at least three months, which is much longer than those obtained for enzyme-based O2˙biosensors .13–17


Typical amperometric responses (insets) and calibration curves of Mn2+/Nafion/TiO2 film toward 1 μM O2˙− at an applied potential of (A) 0.6 V and (B) 0.0 V in 100 mM K3PO4 (pH 7.0).
Fig. 2 Typical amperometric responses (insets) and calibration curves of Mn2+/Nafion/TiO2 film toward 1 μM O2˙ at an applied potential of (A) 0.6 V and (B) 0.0 V in 100 mM K3PO4 (pH 7.0).

The striking advantages of the Mn2+/Nafion/TiO2 film such as relatively long stability and biocompatibility provided a platform to adhere cells directly onto the film surface, and subsequently opened a reliable approach to the detection of O2˙ released from cells. Fig. 3 depicts electrochemical responses obtained at an Mn2+/Nafion/TiO2 electrode adhered with (A) HEK 293T ovary cells and (B) CHO cancer ovary cells with the addition of zymosan, which was reported to generate O2˙ from cells.19 A much higher increased cathodic current was observed from the cancer cells than that obtained from the normal cells. After injection of an SOD solution, a selective scavenger of O2˙ into the solution, the increased currents decreased down to almost background level. Accordingly, the generated increases of cathodic currents are attributed to the reduction of O2˙. The much higher increased cathodic current generated from cancer cells than that obtained from normal cells suggests that O2˙ might be proposed as a biomarker of cancer.


Electrochemical responses of Mn2+/Nafion/TiO2 film toward (A) HEK 293T ovary cells and (B) CHO cancer ovary cells, at an applied potential of 0.0 V after the injection of 10 mM zymosan. The grey bars represent no addition of SOD and the black bars correspond to after the addition of 300 U ml−1 SOD solution.
Fig. 3 Electrochemical responses of Mn2+/Nafion/TiO2 film toward (A) HEK 293T ovary cells and (B) CHO cancer ovary cells, at an applied potential of 0.0 V after the injection of 10 mM zymosan. The grey bars represent no addition of SOD and the black bars correspond to after the addition of 300 U ml−1 SOD solution.

In summary, this communication demonstrates a novel protocol for extremely durable, reliable and selective in situ real-time determination of O2˙ based on direct electron transfer of Mn3(PO4)2, which acts as a SOD. In addition, Mn2+/Nafion/TiO2 film with relatively long stability and biocompatibility provides a platform to adhere living ovary cells directly onto the film surface. The remarkable analytical performance of the present O2˙biosensor , combined with the intrinsic characteristics of the nanostructured TiO2 material provides a new method for the determination of O2˙ released from normal and cancerous ovary cells. This investigation not only demonstrates a novel method to construct third-generation biosensors based on biomimetic enzymes, but also provides a methodology for the determination of O2˙ using this durable and reliable biomimetic probe.

We greatly appreciate Miss Lili Qin at the School of Life Sciences and Technology, Tongji University, for the cell culture and helpful discussion. This work was financially supported by the Program for New Century Excellent Talents in University (NCET-06-0380) and the Scientific Research Foundation for the Returned Overseas Chinese Scholars from State Education Ministry, China, and Shanghai Pujiang Program (06PJ14090). Tongji University is also gratefully acknowledged.

Notes and references

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Footnote

Electronic supplementary information (ESI) available: Details of experiments, electrochemical properties of Mn2(PO4)3 at different matrices; electrochemical reaction process of Mn2(PO4)3 at Nafion-modified TiO2 nanoneedles; selectivity of the present O2˙biosensor . See DOI: 10.1039/b902150g

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