Combination of hematin and PEDOT via 1-pyrenebutanoic acid: a new platform for direct electrochemistry of hematin and biosensing applications

Abstract

In this work, we prepare a novel platform based on poly(3,4-ethylenedioxythiophene) (PEDOT) and 1-pyrenebutanoic acid (PBA). PEDOT is a conductive material of heteroatom doping, which can connect with PBA through π–π stacking. The feasibility of the film is verified via fabricating it on a glassy carbon electrode (GCE); then, hematin is linked with PBA via carboxylate–zirconium–carboxylate coordination bond to prepare a GCE/PEDOT–PBA–hematin biosensor. The electrochemical performance of the biosensor has been tested by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) and current-time curve method (I–T). From CV, a pair of well-defined and quasi-reversible redox peaks, corresponding to the hematin Fe(III)/Fe(II) redox couple, is observed, and the surface coverage (Γ*) of hematin on GCE has been calculated to be 1.2 × 10⁻⁹ mol cm⁻², which is almost 20 times larger than the monolayer coverage of hemin. This value shows that the PEDOT and PBA composite results in a better loading of hematin on the surface of the GCE. In addition, the GCE/PEDOT–PBA–hematin biosensor exhibits strong electro-catalysis activity for H₂O₂ and displays a linear response for the reduction of H₂O₂ in the range of 0.005 to 1.322 mmol L⁻¹ with a detection limit of 0.03 μmol L⁻¹ and a high sensitivity of 2.83 μA mM⁻¹ cm⁻². In addition, the sensor has been applied to the determination of H₂O₂ in real samples, and the response is in the ideal range, which implies that the GCE/PEDOT–PBA–hematin biosensor has promising future applications.

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1 Introduction

Porphyrins have found wide applications as redox catalysts in numerous chemical processes. Hemin, a porphyrins, is the active center of hemoglobin and several other enzymes and is present in proteins in a blood corpuscle.¹ Hematin is a hydroxylated hemin that exhibits strong electrocatalysis to oxygen, peroxide, nitric oxide, carbon dioxide, and hydrogen peroxide.^2–6 The catalytic activities arise from the redox reaction of the ferric/ferrous ions in the center of hematin. However, as a biomimetic catalyst, the catalytic activity and stability of hematin is inferior to natural enzymes.⁷ In recent years, several reports have been dedicated to investigate improvements in the catalytic performance of hematin;^8–11in general, carbon nanotubes or graphene have been used to immobilize hematin. Experiments have indicated that carbon materials can considerably promote the catalytic activities of hematin.^9–11 However, these materials suffer from drawbacks such as cumbersome preparation methods, indirect detection procedures, etc.

In 2001, Chen et al., proposed a method for immobilizing proteins and small biomolecules on carbon nanotubes by 1-pyrenebutanoic acid (PBA). PBA contains four benzene rings, and it is well-known that PBA can interact strongly with carbon nanotubes and graphene via π–π stacking.^12,13 Many studies have focused on methods of immobilizing various enzymes and proteins on carbon materials via PBA.^14–17 However, carbon nanotubes and graphene are expensive, their preparation is also complicated. Aiming at a simple and stable base to immobilize hematin, we have tried to find a material that can connect with PBA via π–π stacking. PEDOT is one of the most studied conducting polymers:^18,19 its good stability, high-speed electron transfer, and easy formation of a tenacious film make it a good material for sensors. First, PEDOT is a conductive material of heteroatom doping and can act as a versatile building block for advanced functional π-conjugated systems because of the amounts and geometry of benzene rings contained in PEDOT.²⁰ Based on the abovementioned considerations, for the first time, we have used PEDOT along with PBA for fabricating a new type of hematin electrochemical biosensor. In this approach, PBA is irreversibly adsorbed on the hydrophobic surfaces of PEDOT film via π–π stacking, and carboxyl groups in PBA are introduced on a glassy carbon electrode (GCE) surface that facilitates the connection of hematin via the coordinate bond of carboxylate–zirconium–carboxylate.^21–24 The association of hematin to PBA is illustrated in Scheme 1.

Scheme 1 Mimetic diagram of the preparation of the electrochemical sensor. (A) Polymerize EDOT to the surface of GCE; (B) PBA connects with PEDOT via π–π interaction; (C) hematin to the functionalized film through carboxylate–zirconium–carboxylate. (red: O; dark yellow: S; gray: C; yellow: Zr; blue: N; brown: Fe; orange ring: PBA).

In general, H₂O₂ is involved in several biological processes. H₂O₂ participates in a wide range of enzymatic reactions. In particular, it plays an important role in biological processes such as the metabolism of proteins and carbohydrates and in immune responses;²⁵ in addition, it is associated with diagnostic response in several biochemical methods for monitoring blood glucose.²⁶ H₂O₂ is also utilized in many industrial processes such as pharmaceutical manufacturing, treatment of paper, textiles and food.^27,28 Therefore, the analysis of H₂O₂ is essential in clinical and industrial samples. Here, we report a new sensitive, biomimetic sensor based on PEDOT, PBA and hematin for H₂O₂ detection.

2 Results and discussion

2.1 Characterization of GCEs with different modifications by XPS and SEM

The characterization of GCE/bare, GCE/PEDOT, GCE/PEDOT–PBA and GCE/PEDOT–PBA–hematin was performed by XPS and SEM. All the XPS spectra were recorded after Ar ion gas etching for 50 s and corrected using a C1s peak at 284.6 eV as an internal standard. Fig. 1 shows the XPS and SEM images of these electrodes: (a) GCE/bare; (b) GCE/PEDOT; (c) GCE/PEDOT-PBA; and (d) GCE/PEDOT–PBA–hematin. As compared to Fig. 1(2)(a), Fig. 1(2)(b) displays surface topography with high roughness and loose structures. From the results of XPS (Fig. 1(1) (A) and Fig. 1(1) (A1)), S peak and increased O peak were obvious. Therefore, SEM and XPS together demonstrate that the PEDOT has been successfully deposited onto the surface of GCE. As shown in Fig. 1(1) (A)(c), the same elements (C, O, S) were detected as that in Fig. 1(1) (A)(b) because PBA is composed of C, H and O. From the SEM image (Fig. 1(2)(c)), we can deduce that PBA was connected with PEDOT through π–π stacking, and its surface density was higher than GCE/PEDOT. As shown in Fig. 1(2)(d), GCE/PEDOT–PBA–hematin renders a rough and dense film with numerous protrusions that could be assigned to the deposition of hematin molecules with large aggregations. In addition, Zr, N and Fe were observed from the XPS of GCE/PEDOT–PBA–hematin; hence, it is confirmed that hematin was linked with PBA. By integrating the results of SEM and XPS, we can find that a new GCE/PEDOT–PBA–hematin sensor is formed as expected.

Fig. 1 XPS spectra (1) and SEM images (2) of different modified GCEs: (a) GCE/bare; (b) GCE/PEDOT; (c) GCE/PEDOT–PBA; (d) GCE/PEDOT–PBA–hematin, (A1) S peak; (A2) Zr peak; (A3) N peak; (A4) Fe peak.

2.2 EIS and CVs of different modified GCEs

Electrochemical impedance spectroscopy (Fig. 2A) and cyclic voltammograms (Fig. 2B) were carried out in 50 mmol L⁻¹ Fe(CN)₆^3−/4− aqueous solution as the electro-active probe using 0.1 mol L⁻¹ KNO₃ as the supporting electrolyte and a saturated calomel electrode as the reference electrode (the same as below). EIS can provide more detailed information regarding the interfacial properties of the surface-modified electrode. Fig. 2A shows the impedance responses of the GCE modified with different layers; an open circuit potential of 0.19 V was selected with frequency ranging from 10⁻² Hz to 10⁵ Hz for the EIS experiments. In general, there are two parts of the result observed on an EIS plot: the first is a semicircular graph found at higher frequencies, which is related to the electron transfer-limited processes in a film and the second is a linear graph found at lower frequencies, resulting from the diffusion-limited processes in a film.²⁹ The diameter of the semicircle exhibited the electron transfer resistance of the layer, showing its blocking behavior for the interface properties of the electrode.

Fig. 2 (A) Electrochemical impedance spectroscopy and (B) cyclic voltammograms of CGE/bare (a); GCE/PEDOT (b); GCE/PEDOT–PBA (c); GCE/PEDOT–PBA–hematin (d); GCE/hematin (e) in 50 mmol L⁻¹ Fe(CN)₆^3−/4− aqueous solution as the electro-active probe with 0.1 mol L⁻¹ KNO₃ as the supporting electrolyte. (C) Modified Randle's equivalent circuit of EIS. Potentials vs. Hg/HgCl/saturated KCl reference electrode.

The impedance data were fitted with a commercial software Autolab data analysis (Metrohm, Switzerland). A modified Randle's equivalent circuit,^30,31 shown in Fig. 2C, was found to fit the data adequately over the entire measured frequency range. The circuit includes the following three elements: (i) the ohmic resistance of the electrolyte solution, R_S; (ii) CPE, associated with the double layer, reflecting the interface between the assembled film and the electrolyte solution; (iii) R_P, the electron transfer resistance.³² Ideally, R_S represents the properties of the electrolyte solution and the diffusion of the redox probe and is not affected by modifications occurring on the electrode surface.³³ A negligible change in R_S was observed during the deposition of PEDOT, the immobilization of PBA, or the coupling of hematin in the last procedure. At the same time, as can be seen in Fig. 2A, the changes in R_P were more significant than those in the other impedance components. Thus, R_P was a suitable signal for sensing the interfacial properties of the GCE during all of the assembly procedures. The fitting values for the stepwise assembled layers on the electrode are presented in Fig. 2C. For the bare GCE, the value of R_P is 134 Ω with a short diameter of the semicircular plot (Fig. 2A(a)), which is a typical characteristic of a diffusion limited electron transfer process.³⁴ When EDOT was polymerized on the GCE, the plot changed to an effective linear relationship because of the fast electron transfer rate of PEDOT (Fig. 2A(b)) with a decrease in the R_P value to 19.7 Ω. A slight increase in the R_P value (to 36.5 Ω) was observed after the deposition of PBA to the PEDOT layer, and the R_P value increased to 70.4 Ω in the successive step of the immobilization of hematin, which may result from the increase in the thickness of the modification on GCE. When compared with GCE/hematin (Fig. 2A(e)), the R_P value of GCE/PEDOT/PBA/hematin decreased nearly 10 times as compared to that of GCE/hematin due to PEDOT, which plays a promoting role in electron transfer. Here, PEDOT immobilized on the electrode played an important role similar to an electron-conducting tunnel, facilitating electron transfer to the electrode surface. These results were consistent with those obtained by CV measurements.

CV is another essential method for studying the interface properties of the modified electrodes. The [Fe(CN)₆]^3−/4− complex ions are electrochemical probes commonly utilized for the investigation of the construction of complex biosensors.³⁵ Well-defined oxidation and reduction peaks of [Fe(CN)₆]^3−/4−(Fig. 2B(a)) were observed for the bare GCE. After the EDOT polymerized on the electrode, the peak current at the modified electrode increased (Fig. 2B(b)), which was attributed to the good conductivity of the PEDOT facilitating electron transfer between the conductive polymer and the electrode surface. When the GCE/PEDOT-PBA (Fig. 2B(c)) and GCE/PEDOT–PBA–hematin (Fig. 2B(d)) were tested, it was found that the catalytic rate of the latter GCE is higher. In contrast, when the GCE was treated merely with hematin, the reversibility decreased as compared to the bare GCE (Fig. 2B(e)). This demonstrated that hematin could impede the electron transfer between the bio-molecules and electrode surface to some extent. However, when both hematin and PEDOT were deposited on the GCE, the cathodic and anodic peak currents clearly increased (Fig. 2B(d)), which can be attributed to the good electrical conductivity of PEDOT.

(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

2.3 Catalytic effect of H₂O₂ on different modified electrodes

To investigate the performance of the five different types of electrodes, cyclic voltammetry experiments were conducted at each electrode immersed in 0.08 mmol L⁻¹ H₂O₂ in 0.1 mol L⁻¹ PBS buffer (pH 7.0), which was stirred and saturated by bubbling N₂ gas. Fig. 3 shows the typical CVs of different modified electrodes. There are no redox peaks for GCE/bare (Fig. 3(a)), GCE/PEDOT (Fig. 3(b)) and GCE/PEDOT–PBA (Fig. 3(c)). When the GCE was modified with hematin, an obvious cathodic peak was observed (Fig. 3(d) and (e)), indicating that the observed redox response is due to the presence of hematin. As shown in Fig. 3, GCE/hematin only yielded a negligible current response after the addition of 0.08 mmol L⁻¹ H₂O₂, indicating that the direct electron transfer between hematin and electrode is difficult. An obvious increase in current is observed in GCE/PEDOT–PBA–hematin as compared to GCE/hematin, which was 3 times larger than that of GCE/hematin. The different performances of the electrodes are mainly attributed to the excellent properties of PEDOT. These conductive materials of heteroatom doping individual sheets have good electrocatalytic activity towards H₂O₂, and the high surface area-to-volume ratio is favorable for hematin immobilization.

Fig. 3 Typical Cyclic voltammograms to H₂O₂ on different modified GCEs in 0.1 mol L⁻¹ PBS (pH 7.0), which contains 0.08 mmol L⁻¹ H₂O₂: (a) GCE/bare; (b) GCE/PEDOT; (c) GCE/PEDOT–PBA; (d) GCE/PEDOT–PBA–hematin; (e) GCE/hematin. Potentials vs. to Hg/HgCl/saturated KCl reference electrode.

Earlier works have suggested that catalysis involves a ferric/ferryl redox cycle (Scheme 2). These circular reaction systems produce both alkoxyl (LO˙) and peroxyl (LOO˙) radical species.³⁶ Step 1: hematin reacts with (H₂O₂) to form ferryl hematin along with various radical species capable of oxidizing substrates such as the heme moiety or the protein. Step 2: protonation of the oxyferryl species to form [Fe⁴⁺−OH⁻]³⁺ destabilizes the complex, giving it a radical-like characteristic. The box defined by the broken line depicts the protonated ferryl species and electronically equivalent radical species. Step 3: auto-reduction of the protonated species occurs by the abstraction of an electron, probably from the porphyrin ring.³⁷

Scheme 2 Mechanism of peroxide-induced ferryl formation and subsequent auto-reduction.

2.4 Effect of scan rate on the direct electrochemistry of hematin on GCE/PEDOT–PBA–hematin

The CVs of the modified electrodes at different scan rates are shown in Fig. 4. The peak current increased as a function of scan rate. The cathodic and anodic peak currents increased linearly with the scan rate from 20 to 300 mV s⁻¹. For the anodic peak current: I = 3.823C − 0.035, R = 0.998; for the cathodic peak current: I = −3.802C + 0.034, R = 0.999. It is clear that hematin was adsorbed on the surface and underwent a surface confined electron transfer;³⁸ its surface coverage (Γ^*) can be calculated according to the Laviron equation:³⁹
where I_p is the peak current, N is the number of electrons transferred (N = 1), F is the Faraday constant, ν is the scan rate, A is the effective surface area (0.07 cm²), Q is the quantity of charge (8.19 × 10⁻⁶ C), R is the gas constant and T is the temperature (298.15 K). From this equation, the surface coverage (Γ^*) was calculated to be 1.2 × 10⁻⁹ mol cm⁻², which is larger than the theoretical monolayer coverage of Hb (6.98 × 10⁻¹¹ mol cm⁻²) and is almost 20 times larger than the monolayer coverage of hemin.^40,41 This value shows that the PEDOT film and PBA lead to better loading of hematin on the surface of the GCE, which enhances the electron transfer rate and catalytic ability of hematin.

Fig. 4 (A) Cyclic voltammograms of GCE/PEDOT–PBA–hematin in 0.1 mol L⁻¹ PBS (pH 7.0) with scan rates of 20–300 mV s⁻¹. (B) Plots of cathodic and anodic peak currents versus scan rate. Potentials vs. to Hg/HgCl/saturated KCl reference electrode.

2.5 Effect of pH on the catalysis of GCE/PEDOT–PBA–hematin to H₂O₂

The pH of the electrolyte is important for the performance of the biosensor. Fig. 5 shows the amperometric response of GCE/PEDOT–PBA–hematin at different pH values (pH 5.0 to pH 9.0) in the presence of 0.08 mmol L⁻¹ of H₂O₂. As can be seen from Fig. 5B, the response current decreased from pH 5.0 and reached the minimum value at pH 6.0, increased from pH 6.0 to pH 7.0, thereafter decreased quickly from pH 7.4 to pH 9.0. The maximum catalysis is at pH 5.0. This may be attributed to the proton involved in the electrochemical reaction. In an alkaline environment, the electrochemical reaction becomes more difficult due to the lack of protons. However, in acidic media, hematin on GCE is not very stable, and thus we chose pH 7.0 as the optimal condition.^38,42 As shown in Fig. 5C, the cathodic peak potentials (Epc) shifted negatively with pH increasing from 5.0 to 9.0. The Epc value showed a linear response to pH from 5.0 to 9.0. The slope was about 40 (±2.6) mV per unit pH, which was smaller than the theoretical value of 57.6 mV per unit pH at 18 °C for single-proton coupled and reversible one-electron transfer.⁴³ The potential at pH 7.0 was −0.3 V, which suggested that the groups near the heme iron affected the redox potential.

Fig. 5 (A) Cyclic voltammograms of GCE/PEDOT–PBA–hematin measured at different pH values at 100 mV s⁻¹ in the presence of 0.08 mmol L⁻¹ H₂O₂; (B) change in current for different pH values; (C) the linear variation of potential with pH values. Potentials vs. to Hg/HgCl/saturated KCl reference electrode.

2.6 Electrocatalytic activities to H₂O₂ of GCE/PEDOT–PBA–hematin sensor

The electrocatalytic behavior of GCE/PEDOT–PBA–hematin towards H₂O₂ was investigated. The catalytic reduction of H₂O₂ at the biosensor was examined by amperometry. The typical current–time plot of GCE/PEDOT–PBA–hematin is given in Fig. 6A. The working potential was set at −0.3 V, which was obtained from Fig. 5. The biosensor responded rapidly when H₂O₂ was successively injected and reached a steady state (95% of the maximum value) within 3 s, indicating fast diffusion of the substrate in the hybrid film on the electrode and the high sensitivity of the biosensor. Fig. 6B shows the calibration curve of the amperometric response and the concentration of H₂O₂. The biosensor has a good linear relationship with H₂O₂ in the range from 0.005 mmol L⁻¹ to 1.322 mmol L⁻¹: I (μA) = −0.2C (mM) − 0.004 with a correlation coefficient of 0.999. The detection limit was estimated to be 0.03 μmol L⁻¹ at a signal-to-noise ratio of 3 and the detection sensitivity is 2.83 μA mM⁻¹ cm⁻², which is considerably higher than the hydrogen peroxide biosensors based on HRP, Mb and Hb [Table 1].

Fig. 6 (A) Current–time curve of GCE/PEDOT–PBA–hematin to H₂O₂; (B) linear relationship of current and H₂O₂ concentration; in pH 7.0, 0.1 mol L⁻¹ PBS, with the potential of −0.3 V and the sample time interval of 80 s upon initially successive additions of 0.0099 mmol L⁻¹ H₂O₂ into a stirring PBS solution and then gradually increasing the H₂O₂ concentration. Potentials vs. to Hg/HgCl/saturated KCl reference electrode.

Table 1 Performances of various modified electrodes in the detection of H₂O₂^a

Modified electrode	Linear range (mM)	Detection limit (μM)	km (mM)	Ref.
a Mb: myoglobin; GO: graphene; Hb: hemoglobin; HRP: horseradish peroxide; H-GNs: hemin-graphene; AuNPs: Au nanoparticles; AuNRs: Au nanorods; GOs@Pdop: graphene oxide sheets (GOs) coated by polydopamine; CS: chitosan; PTMSPA: poly(N-[3-(trimethoxysilyl)propyl]aniline; GNRs: gold nanorods; CMCS: carboxymethyl chitosan; He: hematin; PAR: poly-(acridine red).
Mb–GO–Nafion	0.006–0.088	2.5	—	47
ZnO–GNPs–Nafion–HRP	0.015–1.1	9	1.76	48
H-GNs/AuNPs/GCE	0.0003–1.8	0.11	—	49
Nafion/Hb/AuNRs–GOs@Pdop/GCE	0.0036–6	2	0.7	50
Au/GS/HRP/CS/GCE	0.005–5.13	1.7	2.61	51
Nafion/HRP/AgNL/GC	0.039–5.2	0.13	0.32	52
HRP/PTMSPA@GNR	0.01–1	0.06	—	53
MWCNT–CS–He/PAR-GCE	0.001–0.01	0.61	—	38
GCE/PEDOT–PBA–He	0.005–1.322	0.03	0.27	This work

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When the concentration of H₂O₂ is higher than 1.322 mmol L⁻¹, a response plateau in the calibration curve is observed, showing the characteristics of the Michaelis–Menten kinetic mechanism.⁴⁴ The apparent Michaelis–Menten constant k^app_m can be obtained from the electrochemical version of the Lineweaver–Burk equation: 1/I_ss = 1/I_max + k^app_m/I_maxC,⁴⁵ where I_ss is the steady-state current after the addition of the substrate, I_max is the maximal current measured under the saturated substrate conditions, and C is the bulk concentration of the substrate. According to the abovementioned equation, the k^app_m value for the enzymatic activity of the GCE/PEDOT–PBA–hematin to H₂O₂ was determined to be 0.269 mmol L⁻¹. Compared to the data obtained for heme proteins immobilized for different devices,^49,52,53 the relatively low value of k^app_m demonstrates the enhancement in the affinity and catalytic activity of hematin grafted to the surface of GCE/PEDOT–PBA. Evidently, the functionalized PEDOT film provides a protective environment for promoting electron transfer. This shows that the immobilized hematin retains its bioelectrocatalytic activity and possesses high biological affinity toward H₂O₂.

2.7 Reproducibility and stability testing of biomimetic sensors

The long-term stability of our fabricated biosensor was also investigated by examining its current response during storage in a refrigerator at 4 °C. The biosensor exhibited no obvious decrease in the current response in the first week and maintained about 92% of its initial value after 5 weeks. The repeatability of the measurement was tested by using the same electrode detecting 0.08 mmol L⁻¹ H₂O₂ for 8 times with a relative standard deviation (RSD) of 3.6%. Five electrodes were treated with the same method to verify the reproducibility of the biosensor by testing H₂O₂ of 0.08 mmol L⁻¹. The obtained results revealed a RSD value of 4.2%, which is acceptable.

2.8 Practical applications of biomimetic sensors

To evaluate the ability of the sensor for routine analysis, the sensor was applied for the determination of H₂O₂ in blood serum samples and a commercial oxidant solution. The actual concentration of H₂O₂ in the oxidant solution was determined by the KMnO₄ titration method and by using the GCE/PEDOT–PBA–hematin biosensor prepared in the experiment.⁴⁶ Results are presented in Table 2. The ratio of the two test facilities toward H₂O₂ in real samples is between 0.8–1.07, which shows that there is very good agreement between the results obtained for the commercial oxidant solution by the proposed sensor and those obtained by the KMnO₄ titration method. The results also show good reproducibility.

Table 2 Results of analysis of H₂O₂ in real samples

Sample	Added (μM)	Found by KMnO4 (μM)	Detected by biosensor (μM)	Ratio of the two recovery
Blood serum	—	0	0	—
	20	21.5	23.1	1.07
	50	48.2	50.2	1.04
	100	103.4	110.4	1.07
Qxidant	—	0	0	—
	20	23.8	18.9	0.80
	50	51.2	52.0	1.01
	100	97.6	96.9	0.99

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3 Conclusions

A novel biosensor was successfully fabricated by modifying GCE with PEDOT, PBA and hematin. This GCE/PEDOT–PBA–hematin biosensor showed a better electrocatalytic activity to H₂O₂ as compared to other enzyme modified electrodes [Table 1]. It could be deduced that the increased catalytic response was due to the enlarged specific surface area of the electrode, and the higher electron transfer rate of the PEDOT; hence, an improved synergistic catalytic effect was observed between the hematin and GCE. The satisfactory performance of the biosensor was attributed to its high sensitivity, good stability and reproducibility, wide linear response range, short response time and high analyte specificity. It is very sensitive and could be used to detect trace amounts of H₂O₂ in the range of 0.005–1.322 mmol L⁻¹. The method proposed in this paper can also be expanded to detect oxygen, peroxide, and nitric oxide.

Acknowledgements

We are grateful to the Nanjing University of Science and Technology for its start-up funding, National Natural Science funding (no. 21345002) for this project.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra05886k

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