Introduction

Glutathione (GSH) is an important non-protein thiol compound in animal and plant cells [1, 2]. GSH plays a very important role in scavenging free radicals, detoxifying, maintaining DNA biosynthesis, maintaining intracellular redox homeostasis, and participating in metabolism [3,4,5]. Abnormal levels of GSH are associated with many diseases, such as cancer, diabetes, Alzheimer’s disease, and human immunodeficiency virus (HIV) [6,7,8]. The establishment of accurate, rapid, and sensitive methods for the detection of GSH has important biological and clinical significance. At present, common methods for detecting GSH mainly include colorimetry, electrochemical methods, high-performance liquid chromatography (HPLC), fluorescence spectrometry, and mass spectroscopy [9, 10]. Among them, fluorescence spectroscopy has the advantages of simple operation, instant detection, high sensitivity, and good selectivity [11]. Compared with a single fluorescence response signal, the ratio fluorescence method has the advantage of effectively overcoming the positive and negative errors caused by various interferences (such as environmental changes, sample background signals, and instrument changes), so the ratio fluorescence method with self-calibration characteristics has attracted more and more attention [12,13,14,15,16].

WS2 is a well-known transition metal disulfide with a graphene-like two-dimensional layered structure. Due to the typical small size effect and boundary effect, WS2 QDs have unique optical properties and good biocompatibility [17,18,19]. WS2 QDs are widely used in the fields of catalysis, optical imaging, fluorescence sensing, and batteries due to their good light stability, easy preparation, low cost, and low toxicity [20, 21]. MnO2 nanosheet is a new class of two-dimensional nanomaterials, which has extensive UV-Vis absorption and high specific surface area, so it is an excellent fluorescence quencher and effective scatterer [22, 23]. MnO2 nanosheets can also be reduced to Mn2+ by GSH without harming the environment or human health and have good biocompatibility [24, 25]. These characteristics have caused MnO2 nanosheets to attract widespread attention in the field of building new sensors [26, 27]. Under incident light, the larger MnO2 nanosheets show two scattering peaks in the fluorescence spectrum. First-order scattering (FOS) is a scattering peak at the incident wavelength; second-order scattering (SOS) is a scattering peak at twice the wavelength of the incident light. In general, the intensity of scattered light is related to the size and number of scatterers in solution [28,29,30]. Fraunhofer’s single slit diffraction law [31] shows that FOS is much stronger than SOS. And we observe that the peak intensities of FOS and fluorescence are quite different, while the peak intensities of SOS and fluorescence are relatively close. Therefore, we combined two different optical signals, fluorescence and SOS, and recorded changes in the fluorescence quenching ability and scattered light intensity of the MnO2 nanosheets caused by GSH induction on a fluorometer to obtain high sensitivity and accurate detection.

In this paper, a ratio method for GSH detection was established by combining the fluorescence of WS2 QDs with the SOS of MnO2 nanosheets (Scheme 1). After adding MnO2 nanosheets to WS2 QDs, the fluorescence of WS2 QDs will be quenched by MnO2 nanosheets through IFE, and SOS is enhanced due to the increase of scatterers (the intensity of SOS is close to the fluorescence before GSH was added). With the addition of GSH, the MnO2 nanosheets were reduced to Mn2+, which caused the fluorescence of WS2 QDs to recover and the SOS of the scatterer to decrease. Therefore, the ratio detection of GSH can be realized by the WS2 QDs-MnO2 system. In addition, the proportional system has also been successfully used for the quantitative analysis of GSH in real water samples and human serum, which proves that the WS2 QDs-MnO2 system has great application potential in GSH sensing.

Scheme 1
scheme 1

Schematic principle of fluorescence/scattered light system based on WS2 QDs-MnO2 for GSH ratiometric detection

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Experimental

Materials and apparatus

Sodium tungstate, sodium dodecyl benzene sulfonate (SDBS), and KMnO4 were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Homocysteine (Hcy), L-cysteine (Cys), serine (Ser), tyrosine (Tyr), glycine (Gly), histidine (His), L-glutathione (GSH), glucose, galactose, and bovine serum albumin (BSA) were purchased from Blue Season Technology Development Co., Ltd. (Shanghai, China). NaCl and other metal salts were purchased from Damao Chemical Reagent Factory (Tianjin, China). Deionized water was used to prepare all solutions. All chemicals and reagents were of AR grade and used as received.

Mettler Toledo pH meter was used to measure the pH values of solutions. The UV-Vis absorption spectra were made on a TU-1901 spectrometer (Purkinje, General). The fluorescence spectra were measured on a F-380 fluorescence spectrophotometer (Gangdong scientific and technological development). Fourier transform infrared (FT-IR) was performed on a TENSOR37 (China) spectrometer. X-ray photoelectron spectroscopy (XPS) analyses were carried out on an ESCALab220i-XL spectrometer. The X-ray diffraction (XRD) patterns were given from Bede-D1 X-ray diffractometer. Transmission electron microscopy (TEM) was measured on a Tecnai F20 microscope. Fluorescence lifetimes were recorded on FLS 980 (UK).

Synthesis of WS2 QDs

The synthesis method of WS2 QDs refers to the previous preparation process and has made some modifications [32]. A total of 0.06 g of sodium tungstate was dissolved in 20 mL of water, adjusted to pH = 6.5 with 0.1 mol L−1 HCl, and 5 mL of a 100-mg mL−1 Cys aqueous solution was added. Ultrasonication for 5 min to make it evenly mixed. Then, the mixture was transferred into a 50-mL poly(tetrafluoroethylene) autoclave and reacted at 220 °C for 24 h. The product was slowly cooled to room temperature, centrifuged at 10,000 rpm for 30 min to remove large particles, and the supernatant was collected. Finally, the supernatant was dialyzing in a 1000 Da dialysis bag to remove unreacted material to obtain a WS2 QDs solution.

Preparation of MnO2 nanosheets

MnO2 nanosheets were synthesized by the previously reported method and some modifications were made [33]. In detail, 3.5 mL of ethanol (99%), 3 mL of SDBS (0.5 mol L−1), 0.25 mL of H2SO4 solution (0.1 mol L−1), and 25 mL of distilled water were mixed together; then, the solution was heated to 95 °C. Slowly add 2 mL of KMnO4 solution (0.05 mol L−1) to the above solution and continue stirring for 40 min. After cooling to room temperature, MnO2 nanosheets were collected by centrifugation and repeatedly washed with distilled water for purification. Finally, the prepared MnO2 nanosheets were freeze-dried into a brown-black powder. MnO2 powder was dispersed in distilled water (0.4 mg mL−1) to obtain a MnO2 nanosheet solution.

Quantum yield measurements

The ratio of the fluorescence intensity to the absorbed light intensity of WS2 QDs determines its quantum yield (QY). Absolute QY measurements were taken in a FLS920 spectrometer equipped with a calibrated integrating sphere. The cuvette containing the WS2 QDs aqueous solution was placed in an integrating sphere to measure its QY, and the solvent water was used as a blank sample for reference measurement.

The detection of GSH with WS2 QDs-MnO2 system

First, 100.0 μL of WS2 QDs and 300.0 μL MnO2 solution were placed in a 10-mL volumetric flask, and then, different concentrations of GSH solution (2 mM) were added. The mixture was diluted to 10.0 mL with HEPES buffer at pH = 7. Whereafter, the mixture was incubated at room temperature for 30 min. Finally, the fluorescence emission spectrum was recorded using 320-nm excitation wavelength (5/5 nm slit width).

Results and discussion

Characterizations of WS2 QDs and MnO2 nanosheets

The characteristics of WS2 QDs were analyzed by TEM, FT-IR, XRD, XPS, and fluorescence spectra. Figure 1 a shows the TEM image and particle size distribution of WS2 QDs. It can be seen that WS2 QDs were quasi-spherical and monodisperse, with an average particle size of about 2.3 nm. The FTIR spectrum of WS2 QDs is shown in Fig. 1b. The bending vibration peak of C–H is at 890 cm−1, the stretching vibration peak of C–O is at 1120 cm−1, and the peaks at 1634 cm−1 and 1403 cm−1 are associated to the stretching vibration of C=O and C–N of amide bond. At 3436 cm−1, there are N–H/O–H stretching vibration peaks. The above results indicate that –OH, –NH2, and –COOH exist on the surface of WS2 QDs. Figure 1 c shows that WS2 QDs have characteristic XRD peaks at 2θ = 18.5°, 29.6°, and 50.3°, which correspond to the diffraction peaks of the (002), (100), and (105) lattice planes of WS2 reported in the literature. The XPS method was used to study the surface element composition. As shown in Fig. 1d, the surface of WS2 QDs contains carbon (C), nitrogen (N), oxygen (O), sulfur (S), and tungsten (W) elements. Figure 1 e shows the UV-Vis absorption spectra and fluorescence spectrum of WS2 QDs. The optimal excitation and emission wavelengths of WS2 QDs are 320 and 406 nm, respectively. The WS2 QDs solution is colorless under white light and emits bright blue light under 365 nm UV light (Fig. 1f illustration). The fluorescence emission wavelength changes with the excitation wavelength, indicating that the emission spectrum of WS2 QDs is affected by the excitation light. The measured QY of WS2 QDs aqueous solution was 10.1%, which was higher than the QY of WS2 QDs prepared by other methods.

Fig. 1
figure 1

a TEM image of WS2 QDs(inset) corresponding size distribution histogram of WS2 QDs. b FTIR spectrum of WS2 QDs. c XRD spectrum of WS2 QDs. d XPS spectrum of WS2 QDs. e UV-Vis absorption and fluorescence spectrum of WS2 QDs. f Excitation-dependent fluorescence emission spectra of WS2 QDs, whereas inset shows the blue emission of WS2 QDs under 365-nm excitation

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The characterizations of MnO2 nanosheets were investigated by TEM, UV-Vis, FT-IR, and XRD spectra. TEM microscopy (Fig. 2a) showed that the MnO2 nanosheets had a uniform plate-like structure with occasional folds and crinkles, with an average size of about 300 nm. In addition, as shown in Fig. 2b, the UV-Vis absorption spectrum of the MnO2 nanosheets shows a broad absorption peak in the range of 250 nm to 600 nm, and 380 nm is its characteristic absorption peak. As shown in Fig. 2c, the characteristic peak at 558 cm−1 in the FTIR spectrum of the MnO2 nanosheets is due to the tensile vibration of Mn-O. The XPS spectrum of MnO2 nanosheets is shown in Fig. S1. The XPS spectrum of Mn2p (Fig. 2d) shows two peaks at 642.0 and 653.9 eV, corresponding to Mn2p3/2 and Mn2p1/2 of MnO2, respectively, which are consistent with those reported in the previous literature [34]. The above results indicate the successful formation of MnO2 nanosheets.

Fig. 2
figure 2

a TEM image of MnO2 nanosheets. b UV-Vis absorption of MnO2 nanosheets. c FTIR spectrum of MnO2 nanosheets. d XPS spectrum of Mn2p in MnO2 nanosheets

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Optimization of experimental conditions

In order to develop a colorimetric assay for GSH detection with excellent performance, experimental conditions such as pH, incubation time, and volume of MnO2 nanosheets were optimized.

As shown in Fig. 3a, the F406/S648 values (F406 and S648 are the fluorescence of WS2 QDs at 406 nm and the SOS of system at 648 nm, respectively) are gradually increasing in the pH range of 3.0 to 7.0. Then, as the pH (7.0–10.0) increases, F406/S648 gradually decreases. The maximum is reached at pH = 7.0. In view of the above results, HEPBS buffer (10 mM) having a pH of 7.0 was selected as the working pH of the GSH assay. As shown in Fig. 3b, when GSH was added to the WS2 QDs-MnO2 system, the F406/S648 value increased as the reaction time increased from 0 to 30 min. However, when the reaction time exceeded 30 min, F406/S648 showed almost no change. Therefore, 30 min was selected for further sensitive detection of GSH in further experiments. The volume of MnO2 nanosheets has a great impact on the sensitivity and linear range of GSH detection. As shown in Fig. 3c, as MnO2 was added from 100.0 to 300.0 μL, ΔF (black) and ΔS (red) increased rapidly. ΔF is the change in fluorescence intensity of WS2 QDs at 406 nm in the WS2 QDs-MnO2 system with and without GSH. ΔS is the change in the fluorescence intensity of SOS at 648 nm in the WS2 QDs-MnO2 system with and without GSH. As the volume of MnO2 increases further, they gradually decrease. Therefore, 300.0 μL of MnO2 nanosheets was used as the optimum conditions for subsequent experiments. The fluorescence and SOS spectra of the system at different pH, reaction time, and MnO2 volume are shown in Figs. S2a, S2b, and S2c, respectively.

Fig. 3
figure 3

a Effects of pH to the value of F406/S648. Conditions: MnO2 nanosheets, 300.0 μL; GSH, 60.0 μM; 25 °C for 30 min. b Effects of reaction time to the value of F406/S648. Conditions: MnO2 nanosheets, 300.0 μL; GSH, 60.0 μM; pH = 7.0; 25 °C. c Effects of MnO2 volume to ΔF (black) and ΔS (red). Conditions: GSH, 60.0 μM; pH = 7.0; 25 °C for 30 min. Error bars are the standard deviation of three independent experiments

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Detection strategy and mechanism discussion of GSH

As shown in Fig. 4a, due to the small size of the WS2 QDs, the WS2 QDs that exist alone have only weak SOS signals. In addition, the strength of F406 and S648 was relatively close before GSH was added to the WS2 QDs-MnO2 system. However, when GSH was added, F406 increased and S648 decreased. Therefore, the above phenomenon indicates that the ratio sensing platform can be used for GSH detection.

Fig. 4
figure 4

a Fluorescence and SOS spectra of WS2 QDs-MnO2 (black), WS2 QDs-MnO2 + GSH (red) and WS2 QDs (blue). b UV-Vis absorption spectra of WS2 QDs, MnO2, and WS2 QDs-MnO2. c Excitations and emission spectra of WS2 QDs; UV-Vis absorption spectra of MnO2 nanosheets without or with GSH. d Fluorescence decay curves of WS2 QDs before and after the addition of MnO2 nanosheets

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Next, the mechanism of fluorescence quenching of WS2 QDs by MnO2 nanosheets was studied. Figure 4 b shows the UV-visible absorption spectra of WS2 QDs, MnO2, and WS2 QDs-MnO2. Compared with the absorption spectrum of WS2 QDs alone, there is no new absorption peak after the addition of MnO2, indicating that no non-luminous ground state complex is formed between WS2 QDs and MnO2. So, the static quenching mechanism is excluded. As shown in Fig. 4c, the excitation and emission spectra of WS2 QDs overlap with the UV-visible absorption spectrum of MnO2. Based on the above results, fluorescence resonance energy transfer (FRET) or IFE may occur. In addition, in the absence of MnO2, the fluorescence lifetime of WS2 QDs is 6.16 ns, and the fluorescence lifetime (5.93 ns) hardly changes after adding MnO2, as shown in Fig. 4d and supporting information Table S1. Therefore, the FRET mechanism is excluded from the fluorescence lifetime data, and the results indicate that IFE is the quenching mechanism of WS2 QDs.

GSH has a strong reducing ability. When GSH is added to the WS2 QDs-MnO2 system, GSH reduces MnO2 to Mn2+, resulting in dissociation of MnO2 nanosheets, as shown in the equation 1. At the same time, the disappearance of the characteristic absorption band of MnO2 (Fig. 4c) inhibited the IFE to WS2 QDs, resulting in the fluorescence recovery of WS2 QDs. The reduction in size of MnO2 nanosheets after being decomposed by GSH results in a decrease in the SOS of the system. Therefore, the ratio detection of GSH was obtained by the fluorescence and SOS dual signal response.

$$ 2\mathrm{GSH}+{\mathrm{Mn}\mathrm{O}}_2+2{\mathrm{H}}^{+}\to \mathrm{GSSG}+{\mathrm{Mn}}^{2+}+{\mathrm{H}}_2\mathrm{O} $$
(1)

Ratiometric detection of GSH

In order to prove the applicability of this ratio sensing platform for GSH detection, we investigated the response of the WS2 QDs-MnO2 system to GSH. As shown in Fig. 5a, under the optimal experimental conditions, as GSH concentration increased, F406 increased, and S648 decreased. At the same time, a good linear relationship was obtained over a concentration range of 0 to 60 μM with a correlation coefficient of 0.995 (Fig. 3b). The linear equation is F406/S648 = 0.0161c + 1.169. The LOD is 0.12 μM. In addition, our sensing platform was compared with previously reported strategies for GSH detection (Table 1). The results show that our sensing platform has high sensitivity, wide linear range, and low detection limit. Therefore, the WS2 QDs-MnO2 system shows the potential advantages of determining GSH in actual samples.

Fig. 5
figure 5

a Fluorescence and SOS spectra of WS2 QDs-MnO2 system at different GSH (0–60 μM) concentrations under 320-nm excitation wavelength. b The linear relationship of F406/S648 versus different concentrations of GSH (0–60 μM)

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Table 1 Comparison of reported methods with current work for GSH detection
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The selectivity of GSH detection

Selectivity is an important parameter for studying the performance of fluorescence sensing platforms. In order to evaluate the selectivity of the WS2 QDs-MnO2 system for GSH, the fluorescence responses of WS2 QDs-MnO2 to some metal ions (Na+, K+, Mg2+, Ca2+, Fe3+, and Zn2+), amino acids (Hcy, Cys, Ser, Tyr, Gly, and His), and biomolecules (glucose, galactose, and BSA) were investigated. As shown in Fig. S3, after adding the above interfering substances with the same concentration as GSH (60 μM), the F406/S648 value of the WS2 QDs-MnO2 system was basically unchanged. When GSH was added, the F406/S648 value showed a significant change. Although AA as a reducing agent will cause the F406/S648 value of the system to fluctuate, the concentration of GSH in plasma is much larger than AA, so the effect of AA on the determination of GSH can be ignored. These results show that the WS2 QDs-MnO2 system has good selectivity and competitiveness for the detection of GSH.

Detection of GSH in real samples

In order to evaluate the reliability and applicability of the WS2 QDs-MnO2 system in practical applications, spiked recovery experiments were performed to determine the GSH levels in two actual water samples (tap water and lake water) and human serum. First, the real water sample was filtered through a 0.45 μm membrane to remove large suspended particles, and then, different concentrations of GSH (10, 20, and 30 μM) were added. The WS2 QDs-MnO2 system was used to detect the concentration of GSH in the real water sample after spiking, and each concentration was measured three times. The collected serum sample was centrifuged at 10,000 rpm, and the light yellow supernatant was taken. Because the concentration of GSH in human serum was as high as mM, the supernatant was diluted 50 times with ultrapure water so that the original GSH concentration could fall into the standard calibration curve of this method, and then, the GSH spike recovery experiment was the same. Without spiking, the GSH concentration in the diluted serum sample was 0.56 μM. Tables 2 and 3 list the corresponding analysis results before and after adding GSH to actual water samples and human serum. When we use this platform for determination, the recovery of quantitatively spiked GSH in actual water samples is 95.3–104.1%, and the relative standard deviation (RSD) is 2.29–3.37%. The recovery of GSH in human serum was 95.6–104.3%, and the RSD was 2.79–4.01%. Therefore, the proposed WS2 QDs-MnO2 nanoprobe for detecting GSH in actual water and serum samples is feasible and reliable.

Table 2 Recovery test results of GSH detection in tap water and pool water samples
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Table 3 Recovery test results of GSH detection in human serum samples
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Conclusion

In summary, we have successfully designed a new GSH ratio detection method based on the fluorescence and SOS of the WS2 QDs-MnO2 system. This method has the advantages of simplicity, low cost, wide linear range, high sensitivity, environmental friendliness, and good selectivity. With the addition of large-sized MnO2 nanosheets, the fluorescence of WS2 QDs was quenched by IFE, and the SOS of the scatterer was enhanced to be relatively close to fluorescence. As GSH was gradually added to the WS2 QDs-MnO2 system, GSH reduced the MnO2 nanosheets to Mn2+, which led to the fluorescence recovery of WS2 QDs and the decrease of SOS of the scatterers. In the concentration range of 0 to 60 μM, the value of F406/S648 increased linearly with the increase of GSH, and the LOD was calculated to be 0.12 μM. It is worth noting that the proportional system has been successfully applied to the detection of GSH in actual samples. We believe that the proportional system will have great application potential in the fields of environmental monitoring. This newly proposed ratio detection method provides a new perspective for the construction of new biosensing.