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

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.

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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˙⁻.

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.

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 E^0′ values of the O₂/O₂˙⁻ and O₂˙⁻/H₂O₂ redox couples are −0.35 and 0.68 V vs. Ag|AgCl and thus the E^0′ 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 E^0′ 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

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.

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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