A MnO₂–[Ru(dpp)₃]Cl₂ system for colorimetric and fluorimetric dual-readout detection of H₂O₂

Abstract

Two-dimensional (2D) MnO₂ nanosheets were synthesized by a template-free and one-step route, and the dye [Ru(dpp)₃]Cl₂ was linked onto the MnO₂ nanosheet surface via electrostatic interaction. The formed MnO₂–[Ru(dpp)₃]Cl₂ hybrid was used for a dual optical detection for H₂O₂, an important reactive oxygen species (ROS). Upon addition of H₂O₂, the reaction of MnO₂ with H₂O₂ results in the dissolution of MnO₂ nanosheets and simultaneous generation of O₂. The fading of the solution and simultaneous fluorescence change of [Ru(dpp)₃]Cl₂, sensitive to O₂, enables colorimetric and fluorimetric dual-mode detection of H₂O₂. The dual-output assay in a single probe provides a good sensitivity with a detection limit of 0.18 μM H₂O₂. The dual-signal strategy can efficiently overcome the shortcoming of the single detection mode, and improve the detection accuracy by an additional correction of output signals from each other. Moreover, the successful determination of H₂O₂ in the serum samples demonstrates the potential applicability of the MnO₂–[Ru(dpp)₃]Cl₂ based probe in biosensing and bioanalysis.

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1. Introduction

Hydrogen peroxide (H₂O₂), one of the most important reactive oxygen species (ROS), is present in various biological tissues, and plays critical roles in redox biology and cell signaling.^1–4 Misregulation of H₂O₂ can produce and accumulate oxidative stress inside cells, resulting in damage to biomolecules such as DNA, proteins, and lipids. In the long term, this damage leads to various disorders, such as neurodegeneration, HIV activation, cardiovascular diseases, cancer and aging.^5–10 Also, H₂O₂ is a main intermediate or final product of many enzymatic reactions by a large number of oxidases, thus enabling quantitative assays of the activity of the enzyme as well as various enzyme substrates such as protein and carbohydrate in living organisms via a H₂O₂-mediated process.^11–15 Therefore, the accurate and sensitive detection of H₂O₂ is essential to evaluate its concentration in various biological events and understand the related biological effects.

To date, various analytical techniques have been developed for the detection of H₂O₂, such as high performance liquid chromatography (HPLC) detection,¹⁶ optical sensing,^17–21 colorimetric method,^22–25 electrochemical analysis,^26–28 etc. It is well known that colorimetric and fluorimetric methods are relatively simple, rapid and low cost, but they are of high sensitivity and specificity, and thus they are the typical sensing techniques of optical sensors, which can be easily transformed molecular events into fluorescence intensity or color changes. Colorimetric detection is usually achieved by monitoring the absorbance variation at a specific wavelength induced by the analyte concentration. Moreover, this method permits “naked eye” detection via color change with no requirement for sophisticated instrumentation.^29,30 Fluorimetric methods are performed with the assistance of suitable probes, whose fluorescent signals respond to their interactions with analytes via photo-induced electron transfer (PET), fluorescence resonance energy transfer (FRET) and inner filter effect (IFE).^30–32

It is of great interest to integrate both optical modes into one sensor, by a fluorimetric and colorimetric dual mode strategy, which can minimize the measurement errors and has been proven to be more efficient than the single mode method.³³ Compared with the single-readout analytical strategy, the dual-readout nanomaterials-based protocols endow sensors with improved exactness and increased sensitivity. Commonly, two individual readout probes are combined to realize this dual-readout strategy, which probably complicates the detection process and even leads to unexpected interference. Therefore, a single probe with dual-readout performance was distinctly superior to the combinations of two individual probes in the dual-readout design.^34–37

In this work, two-dimensional (2D) MnO₂ nanosheets were synthesized by a template-free redox route, and the dye [Ru(dpp)₃]Cl₂ was linked onto the surface of MnO₂ nanosheets via an electrostatic interaction. The formed MnO₂–[Ru(dpp)₃]Cl₂ hybrid was used for a colorimetric and fluorimetric detection of H₂O₂, performing the dual-output assay in a single probe. The dual-signal detection strategy in a single probe can hold an additional correction of output signals from each other, thus improving the detection accuracy.

2. Experimental

2.1. Chemicals

Analytical grade KMnO₄, sodium dodecyl sulfate (SDS), concentrated sulfuric acid (H₂SO₄), hydrogen peroxide (H₂O₂) were obtained from Beijing Chemicals Reagents. [Ru(dpp)₃]Cl₂ was purchased from Sigma-Aldrich Co. (Shanghai, China). MilliQ water was used throughout. All other chemical reagents were of analytical reagent grade. The citrate buffer was prepared by mixing an approximate ratio of citric acid and sodium citrate solutions.

2.2. Synthesis of MnO₂ nanosheets

MnO₂ nanosheets were synthesized according to previous publications.³⁸ Typically, 32 mL of SDS solution (0.1 M) and 1.6 mL of H₂SO₄ solution (0.1 M) were added into 283.2 mL distilled water and heated at 95 °C for 15 min. 3.2 mL of KMnO₄ solution (0.05 M) was added into the above solution quickly to start the reaction, and the reaction mixture was maintained at 95 °C for 60 min. In this process, the initial KMnO₄ solution with purplish red color was gradually transformed to the dark brown colloidal suspension. The resulting suspensions were centrifuged, and the precipitates were thoroughly washed with ethanol for 3 times and subsequently dried in air at 50 °C for various analyses. The purified MnO₂ was dispersed in MilliQ water to form a colloidal suspension for analyte detection.

2.3. Preparation of MnO₂/[Ru(dpp)₃]Cl₂ hybrid

A specified concentration (20 μg mL⁻¹) of MnO₂ nanosheets and 50 μM of [Ru(dpp)₃]Cl₂ were added to ethanol, ultrasonicated at room temperature for 30 min and left overnight. Then, they were centrifugated at 12 000 rpm for 15 min to remove the supernatant. The precipitates were dissolved in aqueous solution.

2.4. Characterizations

The X-ray powder diffraction (XRD) data were collected on an X'Pert MPD Philips diffractometer (CuKα X-radiation at 40 kV and 50 mA) with a scanning step of 0.02°. The transmission electron microscopy (TEM) observations were carried out using a JEOL 2200FS microscope. Samples for TEM investigations were prepared by first dispersing the particles in ethanol under assistance of ultrasonification and then dropping one drop of the suspension on a copper TEM grid coated with a holey carbon film. Fourier transform infrared (FT-IR) spectra (Mattson 5000) of the samples were measured in the range of 4000–450 cm⁻¹ in transmission mode. The pellets were prepared by adding 0.8 mg of the sample powder to 80 mg of KBr. The powders were mixed homogeneously and compressed at a pressure of 10 KPa to form transparent pellets. X-ray photoelectron spectroscopy (XPS) analysis was performed using a PHI Quantera SXM (ULVAC-PHI) device operating at a pressure of 10⁻⁸ torr. The photoelectron emission spectra were recorded using a monochromatic Al Kα source (100 W). The angle between the X-ray direction and the emitted electron direction was 45°. The UV-vis absorbance measurements were carried out using a Schimadzu UV-2550 scanning spectrophotometer with a scan rate of 240 nm min⁻¹. The zeta potential measurements were conducted on the same Malvern Nano ZS instrument. The fluorescent response and emission spectrum of the sensor were measured using a 1 cm glass cuvette at 25 °C in citrate buffer (pH 5.6) with a fluorescence spectrophotometer (Hitachi F-4500, Japan) equipped with a xenon lamp. All response curves were acquired with an excitation wavelength of 535 nm. The fluorescence intensity of MnO₂/[Ru(dpp)₃]Cl₂ (10 μg mL⁻¹) at 625 nm as a function of time was recorded by adding 30 μL of H₂O₂ solution to 3 mL of MnO₂/[Ru(dpp)₃]Cl₂ suspensions.

2.5. H₂O₂ detection

H₂O₂ detection was conducted in an open cuvette configuration. In a typical process of H₂O₂ detection, 2 mL of MnO₂/[Ru(dpp)₃]Cl₂ solution (10 μg mL⁻¹) was added to an open cuvette, and a given concentration of H₂O₂ solution was then added slowly. After reaction 5 min, the emission spectrum of [Ru(dpp)₃]Cl₂ was recorded immediately with an excitation of 535 nm.

3. Results and discussion

3.1. Synthesis and characterization of MnO₂ nanosheets and MnO₂/[Ru(dpp)₃]Cl₂ hybrid

A template-free and one-step method was used to synthesize the MnO₂ nanosheets via a redox reaction of KMnO₄ and sodium dodecyl sulfate (SDS). The crystal structure of the synthesized product was characterized by XRD. The XRD profile shown in Fig. 1(a) exhibited four characteristic peaks at 2 theta = 12.1°, 24.2°, 36.7°, 66°, indicating a typical lamellar structure. All the diffraction peaks can be well indexed to δ-MnO₂ phase (JCPDS no. 18-0802). Fig. 1(b) shows a TEM image of representative areas of the as-synthesized product. The sample was typically composed of ultrathin and transparent lamellar structure with ample graphene-like wrinkles and folds, displaying a typical 2D morphology of MnO₂ nanosheets.³⁹ The perceived average lateral dimension of the nanosheets is estimated to be ∼200 nm.

Fig. 1 XRD pattern (a), TEM image (b), FT-IR spectrum (c), XPS spectra of Mn_2p (d) and O_1s (e), and UV-vis absorption spectrum (f) of the synthesized MnO₂ nanosheets.

The FT-IR spectrum (Fig. 1(c)) provides further insight into the structure and surface state of synthesized MnO₂. The peaks at 518, 473 cm⁻¹ are assigned to the characteristic absorption of the Mn–O stretching vibration of octahedral [MnO₆] framework.^40,41 Two intense bands at 3420 and 1625 cm⁻¹ are attributed to the physically adsorbed water and the interlayer water in the MnO₂ nanosheets.⁴⁰ The low intensity of peaks in the 2922–2995 cm⁻¹ region are assignable to the asymmetric and symmetric –CH₂ and –CH₃ stretching due to the use of SDS in the synthesis.⁴²

XPS was used to make a qualitative analysis of chemical valence and binding of the element for the synthesized MnO₂. Two characteristic peaks centered at 642.2 and 654.1 eV correspond to Mn 2p_3/2 and Mn 2p_1/2 of MnO₂, respectively (Fig. 1(d)).^40,43 The spin-energy separation of ∼11.9 eV is also consistent with those previous reports by other research groups.^40,44 No additional signals attributed to Mn₂O₃ and KMnO₄ were found in the XPS spectrum, indicating the generation of pure MnO₂. The XPS spectrum of oxygen exhibits two peaks centered at 529.8 and 532.7 eV (Fig. 1(e)), which are assigned to the lattice oxygen of [MnO₆] octahedra and the oxygen in the interlayer H₂O or H₃O⁺, in good agreement with the FTIR result.⁴⁰

The UV-vis absorption spectrum (Fig. 1(f)) of the synthesized MnO₂ solution exhibits a broad absorption band around ∼375 nm, which is attributed to the d–d transition of Mn(IV) in the octahedral [MnO₆] unit. The wavelength and intensity of the absorbance are in line with the previous findings of single-layer MnO₂ nanosheets by other groups.^45–47 The colloidal suspension of single-layer MnO₂ nanosheets shows good stability and remains stable at 4 °C in the dark for more than 15 days without any precipitates.

The dye [Ru(dpp)₃]Cl₂ was linked onto the MnO₂ nanosheet surface via an electrostatic interaction. In order to verify the interaction between MnO₂ nanosheets and [Ru(dpp)₃]Cl₂, the zeta potential of MnO₂ nanosheets was measured. The zeta potential of MnO₂ nanosheets was 57.2 mV (pH = 7), while it decreased to 36.5 mV as MnO₂ nanosheets were treated with [Ru(dpp)₃]Cl₂. This indicates that there exists an electrostatic interaction, leading to the conjugation of [Ru(dpp)₃]Cl₂ at the surface of MnO₂ nanosheets. This indicates that [Ru(dpp)₃]Cl₂ could be linked onto the MnO₂ nanosheets via an electrostatic interaction.

3.2. Design of dual-signal assay for H₂O₂ in a single probe

The present sensor for H₂O₂ detection consists of MnO₂ nanosheets and an oxygen responsive fluorescent transducer, [Ru(dpp)₃]Cl₂. The as-synthesized MnO₂ nanosheets were firstly incubated with the dye [Ru(dpp)₃]Cl₂, and the dye [Ru(dpp)₃]Cl₂ was linked onto the surface of MnO₂ nanosheets by an electrostatic interaction. Upon the addition of H₂O₂, MnO₂ nanosheets interact with H₂O₂ via a redox reaction following the eqn (1), resulting in the dissolution of MnO₂ nanosheets, which makes the solution fade gradually, enabling the colorimetric detection of H₂O₂. Meanwhile, the in situ generated O₂ in the reaction of MnO₂ with H₂O₂ quenches the luminescence of [Ru(dpp)₃]Cl₂ that is an O₂-sensitive phosphorescent dye.⁴⁸ The changes of oxygen concentration induced by H₂O₂ addition can be transformed into a fluorescence signal using an oxygen responsive transducer, enabling the fluorimetric detection of H₂O₂. Therefore, the present MnO₂–[Ru(dpp)₃]Cl₂ system enables a dual optical detection of H₂O₂, performing the dual readout assay in a single probe.

MnO₂ + H₂O₂ + 2H⁺ = Mn²⁺ + O₂ + 2H₂O (1)

3.3. Optimization of the sensing system

To verify whether the above dual readout sensing principle for H₂O₂ detection is feasible, we conducted a preliminary experiment that a small amount of H₂O₂ was tentatively added to the MnO₂–[Ru(dpp)₃]Cl₂ system. The fading of MnO₂ solution was observed; meanwhile, the luminescence of [Ru(dpp)₃]Cl₂ was decreased, indicating the feasibility of sensing strategy.

The sensing performances can be influenced by several factors, such as reaction pH value, incubation time of MnO₂ and [Ru(dpp)₃]Cl₂ and so on. Therefore, these experimental parameters should be systematically optimized for H₂O₂ detection. The effect of the incubation time of MnO₂ and [Ru(dpp)₃]Cl₂ on H₂O₂ detection was investigated. In the preparation process, the MnO₂ nanosheets were first incubated with the dye [Ru(dpp)₃]Cl₂, and then centrifugated. The incubation time has significant influence on the numbers of [Ru(dpp)₃]Cl₂ molecule linked onto the surface of MnO₂ nanosheets. With the increase of incubation time, the luminescence of [Ru(dpp)₃]Cl₂ increases. Up to 30 min, the luminescence was no longer increased. Thus, the incubation time of 30 min was chosen for the preparation of MnO₂–[Ru(dpp)₃]Cl₂ sample.

The reactivity of MnO₂ with H₂O₂ was found to be strongly dependent on the solution pH value. To demonstrate the effect of solution pH on the reactivity of MnO₂ with H₂O₂, the experiments were conducted as below. The equivalent quantities of MnO₂ were dispersed in 5 mL citrate buffer with different pH values ranging from 4.8 to 6.5 and MilliQ water with pH 7.4, respectively. Then, 50 μL of H₂O₂ solution (100 μM) was immediately added and reacted at room temperature for 3 min. The supernatant was immediately moved to a quartz cuvette for UV-vis absorption measurement. The absorbance of MnO₂ solution with the addition of H₂O₂ decreases remarkably with the decrease of pH value (Fig. 2). For a parallel comparison, we also measured the absorption spectra of MnO₂ solution under different pH conditions, but without the addition of H₂O₂. We can see that the absorbance of MnO₂ solution without the addition of H₂O₂ decreases slightly with the decrease of pH values ranging from 7.4 to 4.8, indicating a slight decomposition of MnO₂ nanosheets under an acidic condition. In contrast, the absorbance of MnO₂ solution with the addition of H₂O₂ decreases remarkably (Fig. 2). The above results indicate that an acidic reaction condition benefits the reaction of MnO₂ nanosheets with H₂O₂, and that the presence of H₂O₂ remarkably accelerates the decomposition of MnO₂ in acidic solution. It is also considered that the speed of O₂ release cannot be too fast during H₂O₂ detection. Here, the citrate buffer of pH 5.6 was chosen for H₂O₂ detection.

Fig. 2 A comparison of the MnO₂ solution absorbance with and without the addition of H₂O₂ under the reaction conditions with different pH values. The reaction time was fixed at 3 min.

3.4. Colorimetric detection of H₂O₂

Fig. 3 shows the UV-vis absorption spectra of MnO₂ solution after reaction upon the addition of various concentrations of H₂O₂ at pH 5.6. All UV-vis absorption spectra exhibited a broad band around 375 nm that originates from the d–d transition of Mn(IV) in the octahedral [MnO₆] unit.³⁸ With an increase of H₂O₂ concentration, the absorbance of the solution decreases gradually due to the reduction of MnO₂ to Mn²⁺ by H₂O₂. Indeed, the variation of color depth of the solution as a result of the change of H₂O₂ concentration added can be observed directly by the naked eyes, as shown in the photographs taken for samples treated with various concentrations of H₂O₂ (inset of Fig. (2)). With the increase of H₂O₂ concentration added, the solution faded gradually. The absorbance exhibits a good linear relationship against H₂O₂ concentration in the range of 0–200 μM. By a linear fitting, the regression equation for H₂O₂ was A = 1.464 − 7370[H₂O₂] (M), where A represents the absorbance of the resulting solution at a given H₂O₂ concentration added. Thus, this colorimetric method provides a simple, low-cost and convenient assay for H₂O₂ since the synthesis of MnO₂ nanosheets is quite simple and this method does not need expensive instruments and complicated operations.

Fig. 3 UV-vis absorption spectra of MnO₂ solution upon the addition of H₂O₂ solution with various concentrations. The inset shows the corresponding photographs of the reaction solution.

3.5. Fluorimetric detection of H₂O₂

3.5.1. Fluorescence response toward H₂O₂. When H₂O₂ was added to the MnO₂–[Ru(dpp)₃]Cl₂ system, the luminescence of [Ru(dpp)₃]Cl₂ sensitive to O₂ was gradually quenched under 535 nm excitation due to the generation of O₂. To know the overall luminescence response process with time after the addition of H₂O₂ to MnO₂–[Ru(dpp)₃]Cl₂, the emission of [Ru(dpp)₃]Cl₂ at 625 nm was monitored continuously in a cuvette by a general fluorometer as a function of time. Fig. 4 shows the time-scanning curves of the emission intensities of [Ru(dpp)₃]Cl₂ at 625 nm upon addition of various concentrations of H₂O₂. It is clearly seen that the luminescence change of [Ru(dpp)₃]Cl₂ depends on time after adding H₂O₂. Overally, the emission intensity of [Ru(dpp)₃]Cl₂ decreased gradually upon time when a certain concentration of H₂O₂ was added to MnO₂–[Ru(dpp)₃]Cl₂ system. The luminescence change is fast at first, and then slows down. Also, the luminescence response is dependent on H₂O₂ concentration. We can see that the increase of H₂O₂ concentration accelerates the response of sensor. Indeed, it is not difficult to understand for time and H₂O₂ concentration dependent fluorescence change. As we know, the luminescence decrease of [Ru(dpp)₃]Cl₂ origins from its luminescence quenching by O₂ produced via the reaction of MnO₂ with H₂O₂. When a certain concentration of H₂O₂ was added, H₂O₂ molecules immediately diffused toward the MnO₂ nanosheet surfaces. At starting, the diffusion of H₂O₂ is fast due to a large driving force as a result of a large concentration gradient, so that more H₂O₂ molecules can reach and interact with the MnO₂ nanosheet surfaces, which generates more O₂ to quench the fluorescence of [Ru(dpp)₃]Cl₂, leading to a higher degree of luminescence change. Upon the depletion of H₂O₂, the luminescence change slows down with time. Similarly, we can also explain H₂O₂ concentration dependent fluorescence response, in that the addition of a higher concentration of H₂O₂ results in a quicker fluorescence response (Fig. 4).

Fig. 4 Time and H₂O₂ concentration dependent luminescence response of [Ru(dpp)₃]Cl₂ under 535 nm excitation.

3.5.2. Stern–Volmer plots. On the basis of the response curves acquired from Fig. 4, the emission spectra of MnO₂–[Ru(dpp)₃]Cl₂ system treated with various concentrations of H₂O₂ were recorded at the time point when the luminescence almost reaches an equilibrium after H₂O₂ addition, as shown in Fig. 5(a). We can see that the emission intensities of [Ru(dpp)₃]Cl₂ at 625 nm decrease gradually upon successive addition of H₂O₂. For a clear comparison, the emission intensity of [Ru(dpp)₃]Cl₂ at 625 nm for MnO₂–[Ru(dpp)₃]Cl₂ treated with various concentrations of H₂O₂ were collected in Fig. 5(b). Clearly, the emission intensity drops rapidly with H₂O₂ concentration over the range of 0–200 μM, indicating a quick quenching.

Fig. 5 (a) The emission spectra of MnO₂–[Ru(dpp)₃]Cl₂ colloidal solution treated with various concentrations of H₂O₂ with 535 nm excitation. (b) The emission intensities of [Ru(dpp)₃]Cl₂ at 625 nm as a function of H₂O₂ concentration. (c) Stern–Volmer plot of H₂O₂ sensing over the range of 0–200 μM H₂O₂. The error bars represent the standard deviation.

The quenching of luminescence in dilute solutions is described by the Stern–Volmer relationship, as shown in the eqn (2).^49–51

F₀/F = 1 + K_SV[Q] (2)

where F₀ is the initial luminescence of MnO₂–[Ru(dpp)₃]Cl₂ system prior to H₂O₂ addition, F is the luminescence of the solution with addition of any given concentration Q of analyte and K_SV is the Stern–Volmer quenching constant. Providing the plot of F₀/F versus Q is linear, K_SV can be determined. Fig. 5(c) shows the Stern–Volmer plot of F₀/F against H₂O₂ concentration. We can see that F₀/F exhibits a good linear relationship against H₂O₂ concentration in the range of 0–200 μM with a correlation coefficient R² = 0.999, indicating a wide linear response for H₂O₂ sensing. From the Stern–Volmer plot, K_SV was calculated to be 22 520 M⁻¹ based on eqn (2) and the linear regression equation for H₂O₂ was F₀/F = 0.99845 + 22 520[H₂O₂] (M). The detection limit (LOD) was defined by the equation LOD = (3σ/s) at the signal-to-noise of 3, where σ is the standard deviation of the blank signals (n = 11) and s is the slope of the calibration curve. Based on this equation, the LOD for H₂O₂ was calculated to be 0.18 μM. The relative standard deviation (RSD) was 4.7% and 3.8% for the determination of 8 μM and 68 μM of H₂O₂ (n = 6), respectively. The above results indicate high reproducibility and reliability of this method for H₂O₂ sensing.

3.5.3. Sensing selectivity. The selectivity of the sensor is essential for detecting an analyte accurately. To demonstrate the sensing selectivity of MnO₂–[Ru(dpp)₃]Cl₂ toward H₂O₂, several bio-active molecules with reducing ability, such as glucose, fructose, glutathione (GSH), and uric acid were used to check whether their presence interfere with the H₂O₂ determination (Fig. 6). Although these molecules with reducing ability reacts with MnO₂ via a redox reaction, no O₂ can release. Therefore, their existence cannot result in the luminescence quenching of [Ru(dpp)₃]Cl₂, indicating high selectivity of our designed sensor toward H₂O₂ detection.

Fig. 6 The luminescence of MnO₂–[Ru(dpp)₃]Cl₂ solution (50 μM) upon the addition of various antioxidants and bio-active metal ions. Na⁺, K⁺, Mg²⁺ and Ca²⁺, 100 μM; Cu⁺, Fe²⁺, 50 μM; glucose, fructose and uric acid, 100 μM; H₂O₂ and GSH, 50 μM. In the vertical coordinate, F/F₀ refers to a ratio, where F₀ is the initial luminescence of MnO₂–[Ru(dpp)₃]Cl₂ prior to any addition, F is the luminescence of the solution with addition of any given concentration of interfering substance.

In addition, several common metal ions in biological fluids, such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu⁺ and Fe²⁺ were added to examine their influence on sensing properties (Fig. 6). The luminescence of [Ru(dpp)₃]Cl₂ was almost unaffected by the extra addition of a certain amount of Na⁺, K⁺, Mg²⁺ and Ca²⁺. Even if several biologically relevant redox active metal ions, such as Cu⁺ and Fe²⁺, were introduced into this sensing system, the luminescence shows only a slight change, compared with the case of added H₂O₂. The above results indicate that the response of our present sensor toward H₂O₂ is highly selective.

3.5.4. Detection of H₂O₂ in biological samples. To demonstrate the practical application of our present sensor, the determination of H₂O₂ in the serum samples was also performed. Three concentrations of H₂O₂ were added into the serum samples. After incubation with MnO₂–[Ru(dpp)₃]Cl₂, the fluorescence signals were detected and analyzed. The H₂O₂ concentrations in the serum samples were obtained according to the linear regression equation, and the resulting results were also listed in Table 1. The recoveries of the H₂O₂ concentration ranged from 92.5% to 112.5%, thus demonstrating the potential applicability of MnO₂–[Ru(dpp)₃]Cl₂ based probe for the quantitative detection of H₂O₂ in biological fluids.

Table 1 Determination of H₂O₂ in serum samples

Sample numbers	Added (μM)	Obtained by our sensor (μM)	Recovery (%)
1	20	22.5	112.5
2	50	48.1	96.2
3	100	92.5	92.5

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4. Conclusions

2D MnO₂ nanosheets were synthesized by a template-free redox route, and the dye [Ru(dpp)₃]Cl₂ was linked onto the MnO₂ nanosheet surface via an electrostatic interaction. The formed MnO₂–[Ru(dpp)₃]Cl₂ hybrid was used for a dual optical detection of H₂O₂. Upon addition of H₂O₂, the fading of MnO₂ solution enables the colorimetric detection for H₂O₂ owing to the decomposition of MnO₂. Simultaneously, the produced O₂ quenches the luminescence of [Ru(dpp)₃]Cl₂, enabling the fluorescent detection of H₂O₂. This sensing system responds linearly and quickly in a wide H₂O₂ concentration range of 0–200 μM, and achieves a detection limit of 0.18 μM and a relative standard deviation lower than 4.7%. The devised dual-readout sensor here could thereby be a reliable option to quantitatively detect H₂O₂ in biological and environmental samples due to additional signal correction from each other, which validated its efficiency in on-site application.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We acknowledge financial support from the National Science Foundation of China (Grant No. 61178073, 61222508).

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