A dual (colorimetric and fluorometric) detection scheme for glutathione and silver (I) based on the oxidase mimicking activity of MnO₂ nanosheets

Abstract

A fluorimetric and colorimetric method is described for the determination of glutathione (GSH) and silver (I). It is based on the use of MnO₂ nanosheets that were prepared by solution mixing and exfoliation. They display oxidase-mimicking activity and can catalyze the oxidation of o-phenylenediamine (OPD) to form yellow 2,3-diaminophenazine (DAP) with an absorption maximum at 410 nm. DAP also has a yellow fluorescence (with a peak at 560 nm). The MnO₂ nanosheets can be rapidly reduced to Mn²⁺ by GSH. This reduces the efficiency of the oxidase mimic MnO₂ and causes a decrease in fluorescence and absorbance intensity. However, on addition of Ag⁺, a complex is formed with GSH. It prevents the destruction of MnO₂ nanosheets so that the enzyme mimicking activity is retained. A dual-method for the determination of GSH and Ag(I) was developed. It has excellent sensitivity for GSH with lower detection limits of 62 nM (fluorimetric) and 0.94 μM (colorimetric). The respective data for Ag(I) are 70 nM and 1.15 μM. The assay was successfully applied to the determination of GSH and Ag(I) in spiked serum samples.

Introduction

The concentration of some life-related small molecules is closely related to disease issues [1, 2]. Therefore, qualitative and quantitative detection can help to provide valuable information for human health [3]. However, owing to the diversity of diseases and the complexity of biological samples, it is still a great challenge to the precise analysis. Therefore, it is necessary to develop more reliable method. The simultaneously detection of two or multiple components is attractive owing to their convenience for realization of multiple purposes [4]. For example, the continuous detection of the copper(II) ion and cysteine (Cys) [5], mercury(II) and Cys [6] have been reported based on the fluorescent nanoprobes. The sensing assays with multiple signals is more selective and sensitive [7]. For example, fluorometric/magnetic bimodal sensor used for the detections of H₂O₂ [8], fluorometric and colorimetric dual-readout for mercury(II) have been reported [9]. Thus, there is considerable interest in developing multi-components detection with multiple signals via the usage of novel functional materials, which can get comprehensive and reliable information.

The rapid development of nanomaterials has brought great opportunities and progress in disease-related fields [10, 11]. Two dimensional nanosheets or nanoflakes have attracted much attention owing to their larger surface area, unique optical features, oxidase or peroxidase mimicking activity [12, 13]. Cobalt oxyhydroxide nanoflakes-carbon dots can monitor ascorbic acid in rat brain [14] and Ti₃C₂ MXenes can detect exosomes in cells [13] based on the fluorescence resonance energy transfer (FRET). V₂O₅ nanosheets with oxidase-like activity can detect glutathione (GSH) in human serum samples [15]. Among the two-dimensional nanosheets, MnO₂ has attracted intense attention because the fascinating characteristics, such as surface property, wide absorption peak, redox properties and biological compatibility [16, 17]. MnO₂-fluorescent polydopamine nanoparticles was applied for alkaline phosphatase (ALP) detection in human serum samples [18], MnO₂-AuNCs can be used for the detection of H₂O₂ based on FRET [8], MnO₂ NFs with tandem enzyme-like activities (GOx-like activity and peroxidase-like activity) can realize the rapid qualitative analysis of glucose by spectrophotometry or visually [19], MnO₂-modified UCNPs can rapidly screen and quantify GSH in cancer cells [20]. To this end, developing MnO₂ based nanoplatform with dual-readouts for multiple components in complex samples is a promising work.

GSH is one of the most typical small molecules, its imbalance is closely associated with numerous clinical diseases, such as cancer, Alzheimer’s disease, liver disease, HIV, heart attack and other ailments [21, 22]. Silver ions (Ag⁺) is one of the hazardous metal ions, which is also highly related to health owing to their bioaccumulation characteristics and widely usage in various fields. Over-used Ag⁺ has brought about a series of problem in environment and health, such as suppressing sulfhydryl enzymes, silver poisoning and silver accumulation [23, 24]. Thus, it is necessary to develop simple and sensitive methods to detect GSH and Ag⁺. A self-cascade catalytic system based on cupric oxide nanoparticles (CuO NPs) was designed for GSH and Ag⁺ ions [25] via intrinsic GSH-oxidase and peroxidase-like activity of CuO NPs. It is effective for detection of GSH and Ag⁺ in serum and tap water, respectively. However, only single channel was utilized, and it is difficult to realize the simultaneously or continuously detection. Thus, it is necessary to develop a simple method with multiple signals, which can be used in the same complex sample for multi-components.

In this work, MnO₂ nanosheets with enzymatic-like activity were prepared and used as the dual-readout nanoprobe for the detection of GSH and Ag⁺ sensitively. As illustrated in Scheme 1, MnO₂ nanosheets can oxidize OPD to DAP with the change of fluorescence and colorimetric signals. GSH can destroy the structure of MnO₂ nanosheets and affect the enzymatic-like property of the MnO₂, which will result in decrease of fluorescence and absorbance intensity of the system. However, when Ag⁺ is added, it can form a complex with GSH [26] and prevent the destruction of MnO₂ nanosheets. Interestingly, the Ag⁺ also can oxidize OPD to DAP [27], to this end, the detection of Ag⁺ via response of fluorescence and absorbance of the system can be realized with increased sensitivity compared with that without GSH. The detection mechanism is thoroughly investigated using various experiments. And it proved that the detection is based on the change of the enzymatic-like properties of MnO₂ nanosheets owing to the damage-protection strategy. The method based on MnO₂-OPD is a dual-readout detection platform for GSH and Ag⁺, which provided more reliable and comprehensive information. The method can detect different components in the same complex sample, which is more convenient and practical. These demonstrate their potential applications in clinical diagnosis and treatment.

Scheme 1

Schematic presentation of the synthesis of MnO₂ nanosheets (a) and the schematic illustration of fluorometric and colorimetric detection of GSH and Ag⁺ on the basis of MnO₂-OPD (b)

Experimental section

Reagents and materials

The single-layer manganese dioxide nanosheets were prepared as literature with slight modification [1]. Tetramethylammonium hydroxide (TMA·OH, 25%) were obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China, http://www.guangfu-chem.com/), o-phenylenediamine (OPD, 99.0%), manganese chloride tetrahydrate (MnCl₂·4H₂O, 99.0%) were purchased from Tianjin Commio Chemical Testing Co., Ltd. (Tianjin, China, http://www.chemreagent.com/), hydrogen peroxide (H₂O₂, 30 wt%) were provided from Shanghai Wokai Biotechnology Co., Ltd. (Shanghai, China, https://ml.hgxqp.com/), L-glutathione reduced (GSH, 98.0%) were purchased from Aladdin industrial corporation (Shanghai, China, https://www.aldjf.com/), bovine serum albumin (BSA, 98.0%) were obtained from Sigma-Aldrich (https://www.sigmaaldrich.com/), di-potassium hydrogen phosphate (K₂HPO_4, 99.5%), potassium dihydrogen phosphate (KH₂PO₄, 99.5%) and silver nitrate (AgNO₃,99.8%) were purchased from Sinopharm chemical reagent Co., Ltd. (Shanghai, China, https://www.reagent.com.cn/). All other chemicals were of analytical grades and used without further purification. Ultrapure water was purified using a Milli-Q system.

Apparatus

Fluorescence spectrometer F-7000 (Hitachi, Japan) was applied for FL analysis. UV–vis spectra were obtained from UV-2450 (Shimadzu, Japan). Fourier infrared spectra (FTIR) were recorded on Nexus-870 (Thermo Nicolet, USA). Energy dispersive spectrometer (EDS) and morphology images of MnO₂ nanosheets were obtained on a transmission electron microscope (TEM) JEM-2100 (JEOL, Japan). X-ray photoelectron spectroscopy (XPS) were obtained from K-Alpha 1063 (Thermo Fisher Scientific, British). X-ray diffraction (XRD) patterns were collected using a Rigaku 2500 (Japan) X-ray diffractometer.

Colorimetric and fluorometric assay of GSH and Ag⁺

A stock solution of GSH (100 mM) was prepared in water and various concentrations of GSH were obtained by serial dilution of the stock solution. For the detection of GSH, 100 μL of GSH solution at different concentrations were added sequentially to 600 μL of 0.1 M PBS (pH 7.0) and 100 μL of MnO₂ nanosheets aqueous solution (200 μM), then 100 μL of 3 mM OPD was added, the total volume of the reaction solution was 1.0 mL. After that, the solution was mixed thoroughly and incubated for 40 min at 60 °C. Finally, colorimetric and fluorescence detection were performed.

The method of detecting Ag⁺ is similar to detecting of GSH. First, 100 μL of 100 μM GSH solution was mixed with different concentration of Ag⁺ (100 μL). Then, the mixed solution was added to 600 μL of 0.1 M PBS (pH 7.0) and 100 μL of MnO₂ nanosheets aqueous solution (200 μM), then added 100 μL of 3 mM OPD, and the total volume of the reaction solution was 1.0 mL. After that, the mixture was mixed thoroughly and incubated for 40 min at 60 °C. Finally, colorimetric and fluorescence detection were performed.

Selectivity

To elevate the selectivity of the method, interferences including common metal ions (Na⁺, Ca²⁺, Mn²⁺, Mg²⁺, Al³⁺, Hg²⁺, Cd²⁺, Cr³⁺_, Pb²⁺), amino acids including glycine (Gly), glutamine (Gln), histidine (His), lysine (Lys), glutamic acid (Glu), tyrosine (Tyr), serine (Ser), alanine (Ala), cysteine (Cys), and other biological compounds like ascorbic acid (AA) that usually coexisted with GSH and Ag⁺ were studied under fluorescence and colorimetric modes.

Detection of GSH and Ag⁺ in real samples

Human serum samples (provided by the Hospital of Hunan Normal University) were treated by centrifugation for 10 min, filtered through a 0.22 μm microporous membrane, and immediately diluted 100 times. Tap water and River water were obtained from our laboratory and the Xiangjiang river located in Changsha, Hunan province, respectively. The water samples were filtered through a 0.22 μm membrane and then diluted 10 times. GSH and Ag⁺ were detected as described above using the standard additional method.

Results and discussion

Characterization of MnO₂

MnO₂ nanosheets were characterized by TEM, UV-vis, FTIR, XRD, XPS and EDS. As illustrated in Fig. 1a, MnO₂ nanosheets exhibit a typical sheet structure with folds and crinkles. MnO₂ nanosheets have a wide absorption band at 300–800 nm with the absorption peak at 380 nm (Fig. 1b) and show dark brown color under sunlight (Fig. 1b, inset). As the FTIR spectra shown in Fig. 1c, MnO₂ nanosheets have characteristic peak at about 500 cm⁻¹ attributed to the Mn-O stretching vibrations. The peaks around 1636 cm⁻¹ may be attributed to O-H bending vibrations combined with Mn atoms, and the peak at 3415 cm⁻¹ suggest the O-H bond stretching vibrations, which are consistent with previous literature [28]. In Fig. 1d, the XRD pattern shows four diffraction peaks located at about 18°, 26°, 36° and 65°, which are indexed as (002), (003), (100) and (110). These suggest the MnO₂ nanosheets has a layered structure as well [29]. In order to further prove the formation of MnO₂ nanosheets, XPS of typical product was recorded (Fig. 1e). The peak of 641.9 eV is fitted into 2p_3/2 and the peaks of 642.4 eV, 641.9 eV and 640.9 eV are fitted into Mn⁴⁺, Mn³⁺ and Mn²⁺, respectively, which accords well with XPS patterns of MnO₂ [30, 31]. EDS in Fig. 1f suggests that the main element is Mn and O. Therefore, the above results confirm the formation of MnO₂ nanosheets.

Fig. 1

a TEM image, b UV-vis spectra (inset shows the photographs of MnO₂ nanoflakes solution under visible light), c FT-IR spectra, d X-ray diffraction pattern, e XPS and f EDS of MnO₂ nanosheets

Feasibility of detecting GSH and Ag⁺

The intrinsic oxidase-like activity of MnO₂ nanosheets was verified. In Fig. S1, MnO₂ nanosheets can catalyze OPD, TMB and ABTS to yellow, blue and green oxidized product, respectively, showing that the MnO₂ nanosheets has oxidase-like activity. Using the typical oxidation of OPD as an example, MnO₂ nanosheets can catalyze the colorless OPD to yellow DAP [16] and lead to the fluorescence and colorimetric signal change. The dual-readout platform may be used for detection of some components that can change the oxidase-like property of MnO₂ nanosheets. Figure 2a, d suggest that the absorbance and fluorescence intensity reduce when GSH is added, but increase in the presence of Ag⁺ for the system of MnO₂-OPD-GSH. As the concentration of GSH increase, the intensity decreases gradually (Fig. 2b, e), while the concentration of Ag⁺ increase, the absorbance and fluorescence intensity enhance gradually (Fig. 2c, f). It is suggested that the fluorescence and colorimetric signal change based on MnO₂-OPD can be used for testing of GSH and Ag⁺ precisely.

Fig. 2

a Absorbance of OPD (a), MnO₂ (b), MnO₂-OPD (c), MnO₂-OPD-GSH (d), OPD-MnO₂-GSH-Ag⁺ (e). Changes in absorbance with different concentrations of GSH (b) and Ag⁺ (c), inset shows the photographs of corresponding solution under visible light. d Fluorescence emission spectra of OPD (a), MnO₂ (b), OPD-MnO₂ (c), OPD-MnO₂-GSH (d), OPD-MnO₂-GSH-Ag⁺ (e). The Changes in fluorescence with different concentrations GSH (e) and Ag⁺ (f). Concentrations for GSH, 0.0 μM (a), 16.7 μM (b), 33.3 μM (c), 50.0 μM (d), 66.7 μM (e); Concentrations for Ag⁺, 0.0 μM (a), 5.0 μM (b), 10.0 μM (c), 15.0 μM (d), 20.0 μM (e)

Mechanism study

To investigate the mechanism of this platform, the TEM, UV-vis spectroscopy, fluorescence spectrum, and XPS were further investigated. As can be seen from the TEM (Fig. 3a), the nanosheets break into smaller nanosheets and Mn²⁺, which is greatly different from the MnO₂ nanosheets. It indicates that the MnO₂ nanosheets may be damaged by GSH. In Fig. 3b, it can be seen that the absorbance intensity decreases, and the color of solution fades (inset of Fig. 3b) with the increasing of GSH concentration. It can be suspected that GSH can reduce the MnO₂ to Mn²⁺ as shown in Eq. (1).

$$ {\mathrm{Mn}\mathrm{O}}_2+2\mathrm{GSH}+{2\mathrm{H}}^{+}\to {\mathrm{Mn}}^{2+}+\mathrm{GSSG}+{2\mathrm{H}}_2\mathrm{O} $$

(1)

Fig. 3

a TEM image of MnO₂ in the presence of GSH. b UV-vis absorption spectrum of MnO₂ nanosheets solution in the absence of GSH (a) and in the presence of GSH with concentrations of 10 μM (b) and 50 μM (c), inset shows the photograph of corresponding solution. c Deconvoluted Mn 2p_3/2 spectra of MnO₂ nanosheets after reduction by GSH. Absorbance (d) and Fluorescence emission (e) of OPD (a), OPD incubated with Ag⁺ (b) and MnO₂ (c), and OPD incubated with the mixture of MnO₂ and Ag⁺ (d). f Fluorescence emission spectrum of OPD incubated with MnO₂ nanosheets (a), GSH incubated with MnO₂ nanosheets prior to addition of OPD (b), OPD incubated with MnO₂ nanosheets prior to addition of GSH (c), GSH was mixed with Ag⁺ firstly, and then the mixed solution incubated with MnO₂ nanosheets prior to addition of OPD (d), GSH incubated with MnO₂ nanosheets prior to addition of Ag⁺, finally OPD was added (e)

In Fig. 3c of XPS, the peak of Mn⁴⁺ on the Mn 2p_3/2 spectrum decreases obviously and the peak of Mn²⁺ increases greatly after the addition of GSH compared with Fig. 1e, which reveals that MnO₂ is reduced to Mn²⁺ by GSH.

In Fig. 3d, e, when Ag⁺ and MnO₂ incubated with OPD, respectively, the absorbance and fluorescence intensity are both enhanced. OPD was added to the mixed solution of Ag⁺ and MnO₂, the intensity is enhanced more clearly. These suggest that both MnO₂ and Ag⁺ make important roles in the oxidation of OPD, and the sensitivity for detection of Ag⁺ can be improved in the presence of MnO₂.

Fluorescence experiment was further applied for proving the mechanism (Fig. 3f). After OPD incubated with MnO₂ nanosheets, the largest fluorescence intensity appeared (a). When GSH incubated with MnO₂ nanosheets prior to addition of OPD (b), the intensity reduced. However, when OPD incubated with MnO₂ nanosheets prior to the addition of GSH (c), there was almost no change in fluorescence intensity compared with MnO₂-OPD. It may suggest that GSH interacts with MnO₂ not OPD. GSH was mixed with Ag⁺ firstly, and then the mixed solution incubated with MnO₂ nanosheets prior to the addition of OPD (d), the intensity was enhanced. However, GSH was mixed with MnO₂ and then added Ag⁺, finally added OPD (e), the fluorescence intensity is almost the same as the case of (b). These results suggest that the mechanism is based on the redox reaction of MnO₂ and GSH, which affects the oxidase property of MnO₂. However, the addition of Ag⁺ will form a complex with GSH and Ag⁺ also can oxidize OPD to DAP, leading to the fluorescence and absorbance intensity enhanced.

Optimization of assay conditions

To obtain the optimal experimental conditions, the pH value, temperature, time and concentrations of OPD, GSH and MnO₂ nanosheets were investigated. pH is a crucial detection condition, the optimal conditions was obtained when the pH value was 7.0 (Fig. S2A, S3A). Reaction temperature influences the reaction speed and stability of reactions, which is also an important for detection. As shown in Fig. S2B and S3B, 60 °C is the optimal reaction temperature. Reaction time also affects the fluorescence intensity, it can be seen that 40 min is the most suitable time for the assay (Fig. S2C, S3C). The concentrations of OPD, MnO₂ nanosheets and GSH are the essential factors in the system, the optimal conditions were obtained when the concentrations of OPD, MnO₂ nanosheets and GSH were 300 μM, 20 μM (Fig. S2E, S3E), 10 μM (Fig. S3F), respectively.

Colorimetric detection of GSH and Ag⁺

Under the optimized conditions, the detection of GSH and Ag⁺ was firstly conducted based on the MnO₂-OPD using the colorimetric method. In Fig. 4a, it can be seen that the absorbance intensity is decreased linearly with the concentrations of GSH and the linear relationship can be expressed as A₀ − A = 0.0016 [GSH] + 0.001 and A₀ − A = 0.0003 [GSH] + 0.0266 in the range of 1.0-20 μM (R² = 0.9914) and 20-80 μM (R² = 0.9916), where A₀ and A represent the intensity of MnO₂-OPD without or with GSH, respectively. The detection limit of GSH is 0.94 μM (S/N = 3) (Fig. 4b). As shown in Fig. 4c, the absorbance intensity is gradually increased with the concentration of Ag⁺. As is exhibited in Fig. 4d, the change of absorbance intensity is closely related to Ag⁺ concentration and it displays good linear relationship as A − A₀ = 0.0026 [Ag⁺] − 0.0288 in the range of 10–70 μM (R² = 0.9907), where A₀ and A represent the absorbance intensity of MnO₂-OPD and GSH without or with Ag⁺, respectively. And the detection limit is 1.15 μM (S/N = 3). Therefore, the platform is suitable for detecting GSH and Ag⁺ based on the colorimetric method using MnO₂-OPD.

Fig. 4

a UV-vis spectra of different concentrations of GSH (0.0, 1.0, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0, 80.0 μM) incubated with MnO₂ nanosheets prior to addition of OPD. b Linear relationship between (A₀ − A) and different concentration of GSH (1.0, 5.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0, 80.0 μM), where A₀ and A represent the intensity of MnO₂-OPD without or with GSH. c UV-vis spectra of GSH and different concentrations of Ag⁺ (0.0, 10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0 μM) incubated with MnO₂ nanosheets and prior to addition OPD. d Linear relationship between (A₀ − A) and different concentration of Ag⁺ (10.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0 μM), where A₀ and A represent the absorbance intensity of MnO₂-OPD-GSH without or with Ag⁺. Inset shows the photograph of corresponding solution

Fluorometric detection of GSH and Ag⁺

Owing to the fluorescence method is more sensitive in the detection, the quantify of GSH and Ag⁺ was also performed. Different concentration of GSH was added in the MnO₂ solution and then OPD was added at optimal assay conditions. In Fig. 5a, it can be observed that the fluorescence signals at 565 nm are sensitive to GSH, and simultaneously, the fluorescence intensity gradually decreases as the concentrations of GSH increasing. The fluorescence signal linearly decreases in the range from 0.5 to 10 μM. As a result, the linear relationship can be described as F₀ − F = 45.905[GSH] − 1.893 with the coefficient of R² = 0.9923 (Fig. 5b), where F₀ and F represent the FL intensities of MnO₂-OPD in the absence or presence of GSH, respectively. and the detection limit is 62 nM (S/N = 3). Similarly, as shown in Fig. 5c, the fluorescence intensity is increased with the concentration of Ag⁺, the linear relationship is F − F₀ = 72.907[Ag⁺] − 0.9923 with the coefficient of R² = 0.9905 (Fig. 5d), where F₀ and F represent the FL intensities of MnO₂-OPD-GSH without or with Ag⁺, respectively. The detection limit is as low as 70 nM (S/N = 3). These consequences demonstrate that the MnO₂-based platform can be applied for GSH and Ag⁺ detection with good sensitivity. The comparison of the performance of this platform with other reported methods for GSH and Ag⁺ are summarized in Table S1. It can be concluded that the detection limit is lower than many other methods. And most of the reports only use single signal or only detecting one component, which is not thoroughly than the dual-readout for multi-components detection.

Fig. 5

a Fluorescence emission spectrum of GSH with different concentrations (0.0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 10.0 μM) incubated with MnO₂ nanosheets prior to addition of OPD. b Linear relationship between (F₀ − F) and different concentrations of GSH (0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, 10.0 μM), where F₀ and F represent the FL intensity of MnO₂-OPD without or with GSH. c Fluorescence emission spectrum of GSH and different concentrations of Ag⁺ (0.0, 0.2, 0.4, 0.6, 0.8, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 μM) incubated with MnO₂ nanosheets and prior to addition of OPD. d Linear relationship between (F − F₀) and the concentration of Ag⁺ (0.2, 0.4, 0.6, 0.8, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 μM), where F₀ and F represent the FL intensity of MnO₂-OPD-GSH without or with Ag⁺

Analytical performance of the method

In order to prove the platform has good selectivity for GSH, some possible interfering molecules including Gly, Gln, His, Lys, Glu, Tyr, Ser, Ala, NaCl, KCl, CaCl₂, MnCl₂, MgSO₄ etc. with the concentration of 100 μM were chosen for study under the same conditions (Fig. 6a) by fluorescence assay because it is more sensitive than colorimetric method. However, large amount of AA and Cys will interfere the detection of GSH owing to its reduction properties which is similar to GSH [32,34,34]. To this end, decreasing the concentration of AA and Cys to 1 μM can eliminate interference since their biological concentration is much lower than GSH. And it is unlikely to cause significant effect on GSH detection in biological systems [1]. It indicated that the platform could selectively distinguish GSH from other species.

Fig. 6

a Selectivity of GSH over potential interferences. The concentrations of GSH (a) was 10 μM, Cys (b) and AA (c) were 1 μM, the concentrations of NaCl (d), KCl (e), CaCl₂ (f), MnCl₂ (g), MgSO₄ (h), Gly (i), Gln (j), His (k), Lys (l), Glu (m), Tyr (n), Ser (o), Ala (p) were 100 μM. b Selectivity of the MnO₂-OPD-GSH toward Ag⁺, the concentrations of Ag⁺ (a) was 10 μM, concentrations of Mn²⁺ (b), Mg²⁺ (c), Hg²⁺ (d), Al³⁺ (e), Ca²⁺ (f), Cd²⁺ (g), Ni²⁺ (h), Zn²⁺ (i), Cu²⁺ (j), Fe²⁺ (n), Fe³⁺ (o), Cr³⁺ (p), Pb²⁺ (q) were 100 μM, concentrations of Na⁺ (k), K⁺ (l), Co²⁺ (m) was 50 μM, and the solution of Cu²⁺ was added EDTA as mask regent. All experiments were performed in pH 7.0 and the error bar represents the standard deviation for three determinations

In order to explore the potential application of Ag⁺ in real samples, we investigated the stability under various ions including Mn²⁺, Mg²⁺, Hg²⁺, Al³⁺, Ca²⁺, Cu²⁺ etc. with the concentration of 100 μM, Na⁺, K⁺, Co²⁺ with the concentration of 50 μM (Fig. 6b). In the system, Cu²⁺ will cause some interference to the detection of GSH, thus EDTA was chose as a masking agent because it could form more stable complexes with Cu²⁺ [2]. Therefore, the platform based on MnO₂ can be used for selectively detection of Ag ⁺.

The method exhibits good selectivity toward GSH and Ag⁺. In order to evaluate the practical performance, human serum samples and water samples were detected (Table 1) using the standard addition method. The recoveries are observed in the range of 90.3–113.2%, further indicating the reliability of this method.

Table 1 The determination of GSH and Ag⁺ in real samples

Conclusions

A fluorometric/colorimetric strategy for sensitive and selective determination of GSH and Ag⁺ based on the MnO₂-OPD nanoplatform was constructed via the damage-protection strategy. The dual-readout detection for GSH and Ag⁺ exhibits good performance with low detection limits and wide linear ranges. The detection mechanism is based on enzymatic properties of MnO₂ nanosheets, which can catalyze the oxidation of OPD into DAP with fluorescence and colorimetric signal change. While upon GSH introduction, the MnO₂ nanosheets can be rapidly reduced to Mn²⁺, which affect its enzymatic properties and result in decrease in fluorescence and absorbance intensity of the system. However, when Ag⁺ is added, it can form a complex with GSH, thus prevent the destruction of MnO₂ nanosheets. And Ag⁺ can catalyze OPD, thus enhancing the fluorescence and absorbance intensity. The efficient preparation of MnO₂ nanosheets, the novel detection mechanism, the excellent applicability in human serum samples and water samples, demonstrate the potential application of MnO₂ nanosheets in clinical diagnosis even early diagnosis of cancer and targeted drugs releasing systems.