Introduction

Glutathione (GSH) is a tripeptide containing sulfhydryl groups that consists of glutamate, cysteine, and glycine. GSH plays critical roles in physiological functions of human body, which has antioxidant, detoxification, metabolism, and intracellular signal transduction features [1, 2]. However, abnormalities of intracellular GSH levels could cause various diseases such as Parkinson disease [3], liver injury [4], Alzheimer disease [5], and acquired immune deficiency syndrome [6]. Thus, it is essential to detect the level of GSH in human to maintain the quality of life. Up to now, studies indicate that there are several methods for detecting GSH, including high-performance liquid chromatography, mass spectrometry [7], electrochemiluminescence [8], surface enhanced Raman scattering [9], enzyme linked immunosorbent assay [10], and fluorescence spectrometry [11]. Compared with the above-mentioned methods, fluorescence spectrometry has unique advantages of high sensitivity, simplicity, rapidity, and low cost in detection. Among them, nanomaterials are often used as the probes to detect analyte based on spectrofluorometric method. Consequently, the emergence of nanomaterials provides a new opportunity of detecting GSH. So far, nanomaterials such as Au nanocubes, up-conversion luminescent materials, and magnetic metal organic framework have been used to detect GSH, of which, Au nanocubes are limited owing to their higher cost and complex synthesis procedures [12]. Up-conversion luminescent materials [13] have the disadvantages of the inherently weak photon response and high toxicity, while magnetic metal organic framework has low sensitivity and narrow linear working range to GSH [14]. Over the years, some researchers have dedicated to investigate new advanced nanomaterials or improve those pre-existing nanomaterials for developing simple, rapid, and sensitive GSH nanoprobes [15]. Compared with the above-mentioned nanomaterials, graphitized carbon nitride (g-C3N4), as a new type of metal-free semiconductor carbon nanomaterials [16], has attracted extensive attention owning to its obvious advantages of stable, optical, and environment-friendly properties. In addition, g-C3N4 has been widely used in the fields of catalysis [17], photoelectrochemistry [18], sensing [19], bioimaging [20], drug delivery [21], and photodynamic therapy [22]. In order to further extend the applications of g-C3N4 in these fields, researchers have adopted the surface modification and functionalization to obtain g-C3N4 with excellent properties, such as higher quantum yield [23], better photostability [24], better biocompatibility [25], and lower toxicity [26].

MnO2 nanosheet, as an emerging 2D-layered material, has attracting extensive attention due to its excellent properties including high specific surface area, better light-absorption, fluorescence quenching capability and excellent biocompatibility [27,28,29]. Yan et al. [27] designed MnO2 nanosheet-carbon dots sensing platform for sensitive detection of organophosphorus pesticides. Xue et al. [30] synthesized an impedance biosensor based on magnetic nanobead net and MnO2 nanoflowers for rapid and sensitive detection of foodborne bacteria. Yuan et al. [31] provided MnO2-nanosheet-modified up-conversion nanosystem to detect H2O2 and glucose in blood. Li et al. [32] fabricated morphology-dependent MnO2/nitrogen-doped graphene nanocomposites for simultaneous detection of trace dopamine and uric acid.

In this work, MnO2-S, O-CNQDs nanocomposite via in-situ synthesis of MnO2 nanosheets in S, O-CNQDs dispersion solution has been prepared. Compared with other fluorescent nanomaterials, the combination of doped carbon nitride with higher quantum yield (23.4%) and manganese dioxide can achieve higher detection sensitivity. The nanocomposite does not show fluorescence due to the fluorescence resonance energy transfer (FRET) between MnO2 nanosheets and S, O-CNQDs. However, when MnO2-S, O-CNQDs interacts with GSH, the fluorescence of S,O-CNQDs is regenerated, attributed to the specific binding of GSH to MnO2. Based on this working principle, a rapid, highly selective and low-toxic MnO2-S, O-CNQDs nanoprobe has been developed to detect intracellular GSH as depicted in Scheme 1.

Scheme 1
scheme 1

MnO2-S, O-CNQDs nanocomposite for GSH assay and reaction mechanism

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Our results indicate that MnO2-S, O-CNQDs is a promising nanoprobe material for quantifying trace amounts of GSH in biological samples as well as monitoring the intracellular level of GSH. It is anticipated that our proposed work could offer a wider application of nanoprobe for multifarious targets via an “off–on” fluorescence detection in biological fields.

Experimental section

Materials and reagents

Thiourea, ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), KMnO4, and 2-(N-morpholino)ethanesulfonic acid (MES) were purchased from Aladdin Co., Ltd. (Shanghai, China). Glutathione, His, Cys, Ala, Thr, Gly, Phe, Trp, Val, glucose, and fructose were bought from Macklin Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Co., Ltd. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM) was acquired from Gibco Co., Ltd. (St. Louis, MO, USA). Both serum samples and fresh urine samples were obtained from healthy volunteers at The First Hospital of Shanxi Medical University and stored at − 20°C. All reagents were used without further purification. All the solutions were prepared using ultra-pure water.

Measurements

The morphology of the as-synthesized materials was characterized by transmission electron microscopy (TEM) using a JEM 2100-H TEM (Shimadzu, Kyoto, Japan). X-ray diffraction (XRD) was measured on a Bruker D8 Advance powder X-ray diffractometer (Bremen, Germany). X-ray photoelectron spectra (XPS) were performed on a Kratos Axis UltraDLD X-ray photoelectron spectrometer (Hadano, Kanagawa, Japan). The Fourier transform infrared spectra (FT-IR) of S, O-CNQDs, MnO2, MnO2-S, O-CNQDs nanocomposite were carried out on a Varian FT-IR-8400 s spectrometer (Agilent Technologies, Santa Clara, CA, USA). The fluorescence and UV–visible absorption spectra were collected on an FLS920 steady-state transient fluorescence spectrofluorometer (Edinburgh Instruments, Livingston, UK) and Metash 6100 UV/vis spectrophotometer (Meipu Instrument, Shanghai, China), respectively. The samples of solid powder were dried and kept on an FDB-3a vacuum freeze dryer (SIM, Los Angeles, USA). The cellular fluorescence images were obtained from an Olympus FV1000 laser scanning confocal microscope (Leica, Wetzlar, Germany).

Preparation of S,O-CNQDs

S, O-CNQDs were synthesized by a solid-phase reaction method [33]. Firstly, 0.3722 g EDTA-2Na and 0.9134 g thiourea were placed and ground into a mortar to obtain the mixture. The as-obtained powder was then transferred to a crucible and heated at 200 °C for 2 h. After cooling to room temperature, the brown colloidal product was washed three times with ethanol. Then, the brown colloidal product was dissolved in 10 mL ultra-pure water to obtain the brownish-red solution. The solution was centrifuged at 8000 rpm to remove the precipitation, and the supernatant was filtered with a 0.22-μm microporous membrane. Finally, the purified S,O-CNQDs was obtained through dialyzing with a dialysis bag (Solarbio, Beijing, China) of 500 Da molecular weight cutoff for 8 h and followed by freeze-dried in vacuum.

Fabrication of MnO2-S,O-CNQDs

Five hundred microliters of S, O-CNQDs (3 mg/mL) was pipetted to the centrifuge tube containing 1.5 mL of MES buffer solution (0.10 M, pH 6.0). Then, 1.0 mL of KMnO4 (15 mM) was dropped into the centrifuge tube and diluted with ultra-pure water to 5.0 mL. Then, the solution was sonicated for 30 min to form a brown colloidal solution. The purified MnO2-S, O-CNQDs nanocomposite was acquired by centrifugation at 8000 rpm for 10 min and washed several times with ultra-pure water. As a control, bare MnO2 nanosheets were synthesized using the similar method without S, O-CNQDs. All the experiments were carried out at room temperature.

Fluorescence sensing of GSH

Various concentrations of GSH (0.0–270 μM) were mixed with a known quantity of MnO2-S, O-CNQDs nanocomposite and ultra-pure water to form 3.0 mL of mixture solution which were kept for 6 min at room temperature. Afterward, the fluorescence emission spectra of the solutions were recorded at an excitation wavelength of 360 nm.

Detection of GSH in human biological samples and glutathione injections

Human serum samples and acetonitrile were mixed with a volume ratio of 1:1. After 3 min, the samples were centrifuged in a 4000 rpm for 30 min to collect the supernatant, which was diluted 100 times with PBS buffer solution (10 mM, pH 6.4) and used for subsequent experiments. The as-prepared samples were mixed with an equal volume of 10 μM, 180 μM, and 250 μM GSH, respectively. Similarly, urine samples were centrifugated to obtain the supernatants which were diluted 1000 times with ultra-water for later use. The rest of the operation was the same as the serum samples. Reduced glutathione injection was a mixture of glutathione and 0.9% sodium chloride injection. Then, the fluorescence spectra of the mixture were measured at an excitation wavelength of 360 nm.

Cytotoxicity assay

The potential cytotoxicity of MnO2-S, O-CNQDs nanocomposite was evaluated by MTT assay with HepG2 cells. The cell suspensions were seeded onto a 96-well plate (5 × 104 cells per well) with 24-h incubation. After the removal of old medium, each well was washed with PBS for several times. Then, adherent cells of each well were cultivated in 500 μL DMEM containing MnO2-S, O-CNQDs nanocomposite at various concentrations (0.0–300 μg/mL) for 24 h. The supernatant was removed, and 20 μL MTT solution (5.0 mg/mL) was injected to each well for 4-h incubation. Afterward, the formed formazan crystals were dissolved in 150 μL DMSO by shaking for 10 min. Similarly, a control group was performed without MnO2-S, O-CNQDs nanocomposite. A blank group was processed in the absence of HepG2 cells and the nanocomposite. The rest operation of the control and blank groups were the same as the experimental group. Finally, the absorbances were recorded through a microplate reader at 490 nm. The cell viability was calculated according to the equation:

$$ {\text{Cell viability (\% )}} = \left( {{\text{OD}}_{s} - {\text{OD}}_{b} } \right)/\left( {{\text{OD}}_{c} - {\text{OD}}_{b} } \right) $$

where ODs and ODc represent the absorbances of the experimental and control group cells at 490 nm, respectively. ODb was recorded in the absence of cells and MnO2-S, O-CNQDs.

Fluorescence imaging of HepG2 cells

HepG2 cells with a density of 5 × 105 cells in each well were incubated in laser confocal dishes under an atmosphere of 5% CO2 at 37 °C for 24 h. The cultured cells were divided into the experimental and control groups. In the experimental group, 200 μg/mL of MnO2-S, O-CNQDs was added into the cells and cultivated for 3 h. Then, the cells were washed with PBS to remove the free MnO2-S, O-CNQDs, whereas the cells of the control group were preincubated with 500 mM N-ethylmaleimide (NEM) for 30 min and washed several times with PBS. The rest of the process was the same as the control group. The fluorescence images of HepG2 cells were performed on a confocal microscope at excitation wavelengths of 405 and 488 nm, respectively.

Results and discussion

Characterization of MnO2-S, O-CNQDs nanocomposite, S,O-CNQDs and MnO2 nanosheets

The TEM images of MnO2, S, O-CNQDs and MnO2-S,O-CNQDs nanocomposite are displayed in Fig. 1.

Figure 1
figure 1

TEM images of a MnO2 nanosheets, b S, O-CNQDs, and c MnO2-S,O-CNQDs nanocomposite

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Figure 1a shows that MnO2 forms a lamellar nanosheet structure. The as-prepared S, O-CNQDs having uniform size and good dispersion with 0.32 nm lattice spacing are depicted in Fig. 1b. Furthermore, Fig. 1c shows that the MnO2-S, O-CNQDs nanocomposite is composed of S, O-CNQDs adhered to the surface of MnO2 nanosheets. The functional groups of MnO2-S, O-CNQDs nanocomposite were identified by FT-IR and are shown in Fig. S1. The absorption peak at 3434 cm−1 is attributed to the stretching vibration of O–H [34]. The absorption peak of S–H group is located at 2120 cm−1, while the peaks at 1670 and 1410 cm−1 are originated from the stretching vibration of C=O and the existence of –COOH [35]. Finally, an absorption peak at ca. 570 cm−1 of MnO2-S, O-CNQDs nanocomposite is identified, corresponding to the Mn–O stretching vibration [36].

The chemical compositions of MnO2-S, O-CNQDs nanocomposite were analyzed by XPS and are depicted in Fig. 2.

Figure 2
figure 2

a XPS full spectrum of MnO2-S, O-CNQDs nanocomposite. High-resolution XPS spectra of b C1s, c N1s, d O1s, e S2p, and f Mn2p

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Figure 2a shows that MnO2-S, O-CNQDs contains C, N, O, S, and Mn elements. The C1s fine spectrum of MnO2-S,O-CNQDs in Fig. 2b exhibits four peaks at 284.90, 285.53, 286.58, and 288.60 eV, attributed to the C=C, C–S, C–O, and C=N bonds, respectively [37]. Figure 2c displays the N 1 s spectrum of C3N4 XPS which indicates the presence of pyridine-type nitrogen (400.10 eV), C-N (398.50 eV), N–(C)3 (399.30 eV), and N–H (400.85 eV) in MnO2-S,O-CNQDs [38]. The fine spectrum of O1s of MnO2-S,O-CNQDs in Fig. 2d shows three characteristic absorption peaks at 530.00, 531.57, and 532.26 eV, corresponding to the binding energies of O–H, C=O, and C–O bonds [39]. The S2p spectrum of MnO2-S,O-CNQDs in Fig. 2e has two characteristic absorption peaks, ascribing to the binding energies of C–S bond at the position of 2p1/2 (163.95 eV) and 2p3/2 (168.85 eV), respectively [34]. Two peaks at 642.27 and 653.86 eV in Fig. 2f belong to the binding energies of Mn2p3/2 and Mn2p1/2 of MnO2-S, O-CNQDs nanocomposite, respectively [40].

Figure 3 depicts the XRD patterns of S, O-CNQDs, MnO2 nanosheets, and MnO2-S,O-CNQDs nanocomposite. There are MnO2 nanosheets with four diffraction peaks at 26.7, 37.2, 45.7, and 56.8° [41]. The diffraction peaks of S,O-CNQDs are centered at 13.4° and 26.8°, corresponding to the in-plane structural packing (100) plane of tri-s-triazine units and the typical graphitic interlayer stacking (002) plane, respectively. The diffraction pattern of MnO2-S, O-CNQDs nanocomposite shows that there are no excrescent diffraction peaks other than the characteristic peaks of MnO2 nanosheets and S, O-CNQDs. In essence, the characterization results demonstrate that MnO2-S, O-CNQDs nanocomposite has been successfully prepared.

Figure 3
figure 3

XRD patterns of S, O-CNQDs (blue), MnO2 nanosheets (black), and MnO2-S, O-CNQDs nanocomposite (red)

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The stability of MnO2-S,O-CNQDs nanocomposite

The stability of MnO2-S, O-CNQDs nanocomposite was studied under different concentrations of NaCl, UV irradiation, and storage. The experiments were conducted in PBS buffer solution with pH 7.0 at room temperature. Figure S2(a) shows that the fluorescence intensity of MnO2-S,O-CNQDs remains almost unchanged under high concentration of NaCl (1.0 M), indicating that MnO2-S,O-CNQDs have high stability in high-ionic-strength medium. After UV irradiation for 180 min, the fluorescence intensity of MnO2-S, O-CNQDs maintains relatively stable as displayed in Fig. S2(b), manifesting that MnO2-S,O-CNQDs has good photobleaching resistance. The fluorescence intensity of MnO2-S, O-CNQDs maintains 95% of its initial intensity after 60 d of storage as depicted in Fig. S2(c). These results indicate that MnO2-S, O-CNQDs nanocomposite possesses good stability which can be a promising fluorescence probe.

The optimization of experimental conditions

In order to obtain a promising fluorescence nanoprobe, the effect of KMnO4 concentrations for preparing MnO2-S,O-CNQDs nanocomposite were investigated at room temperature. Figure S3 displays the fluorescence spectra of MnO2-S, O-CNQDs nanocomposites prepared from various concentrations of KMnO4 and S, O-CNQDs. It is obvious that the fluorescence of MnO2-S, O-CNQDs nanocomposite is gradually quenched with the increase in KMnO4 concentrations. When the concentration of KMnO4 increases to 15 mM, the fluorescence of S, O-CNQDs drops to the lowest. Further increase in KMnO4 concentration does not affect the fluorescence of MnO2-S, O-CNQDs nanocomposite. As such, 15 mM was selected as the optimal concentration of KMnO4 for preparing MnO2-S, O-CNQDs nanocomposite.

Detection of GSH

The reaction time of GSH and MnO2-S, O-CNQDs nanocomposite was optimized. Figure S4 displays the plot of F0/F against t (reaction time) of GSH (50 μM) and MnO2-S, O-CNQDs nanocomposite (5.0 mg/mL), where F0 and F are the initial fluorescence intensity and intensity of MnO2-S, O-CNQDs nanocomposite at t, respectively. It can be observed that F0/F increases rapidly after the addition of GSH and then reaches the highest at 6.0 min. Further increase in reaction time does not increase the F0/F. As a result, 6.0 min was chosen as the optimal reaction time in the subsequent work.

To verify the applicability of our proposed “turn-on” nanoprobe for detection of GSH, the fluorescence of MnO2-S, O-CNQDs at various GSH concentrations was investigated and is shown in Fig. 4. The fluorescence of MnO2-S, O-CNQDs gradually increases with the increase in concentration of GSH. The inset of Fig. 4 shows the linear relationship (r2 = 0.997) between F/F0 and GSH concentration (10–270 μM), where F0 and F are the fluorescence intensities of MnO2-S, O-CNQDs in the absence and presence of GSH. The detection limit is determined to be 0.307 μM (S/N = 3).

Figure 4
figure 4

The fluorescence spectra of MnO2-S, O-CNQDs nanocomposite at various concentrations of GSH. 1–19: 0.0–270 μM. The inset shows the linear relationship between F/F0 and GSH concentration in the range 10–270 μM

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In order to compare with MnO2-S, O-CNQDs, a solution containing MnO2 and S, O-CNQDs was also used for GSH detection. The corresponding results are presented in Fig. S5. When different concentrations of GSH are added to the solutions, the fluorescence of the solutions is restored gradually. The linear relationship (r2 = 0.998) is obtained between F/F0 and the concentration of GSH in the range 20–80 μM with a detection limit of 7.14 μM. Compared to the solution containing MnO2 and S, O-CNQDs, MnO2-S,O-CNQDs shows a wider linear range and lower detection limit. In order to determine repeatability of measurement, 50, 100, and 150 μM GSH are used to determine their F0/F at 10 repeat measurements. The relative standard deviation of the fluorescence intensity of MnO2-S, O-CNQDs is 4.20%, indicating that the nanoprobe possesses good repeatability of fluorescence intensity. Table 1 compares the analytical performance of our proposed nanoprobe with other reported fluorescence nanoprobes for GSH detection. The analytical performance of our nanoprobe is comparable with other fluorescence nanoprobes for GSH detection. In addition, our nanoprobe displays a wide linear range and reasonable detection limit.

Table 1 Different fluorescence nanoprobes for detecting GSH
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Selectivity and interference assays of MnO2-S, O-CNQDs nanocomposite for GSH

In order to evaluate the selectivity and interference of MnO2-S, O-CNQDs nanocomposite toward GSH, the reaction of nanocomposite with the interfering substances including common metal ions, anions, amino acids, and sugars at concentrations of 200 μM under the optimal experimental conditions was investigated and is shown in Fig. 5. Figure 5a shows that MnO2-S, O-CNQDs nanocomposite responds to GSH only, whereas the interferents do not give any significant effect. When GSH is co-exited with 30-fold concentrations of various interferents, the F/F0 shows almost no significant change as depicted in Fig. 5b. Compared to other published nanoprobes for GSH detection, our proposed MnO2-S, O-CNQDs have better selectivity and anti-interference ability. These results indicate that MnO2-S, O-CNQDs nanocomposite could be an ideal “turn-on” probe for selective detection of GSH in the presence of interferents.

Figure 5
figure 5

The selectivity a and interference b of MnO2-S, O-CNQDs nanocomposite for GSH and non-target substances. NAC: N-acetyl-L-cysteine (n = 3)

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Study on the interaction mechanism between MnO2-S, O-CNQDs nanocomposite and GSH

Fluorescence resonance energy transfer is a phenomenon of non-radiative energy transfer between two fluorescence chromophores, which are energy donor and energy acceptor through the dipole–dipole interaction. The process of FRET manifests that the fluorescence lifetime of the donor is shortened and the fluorescence intensity of the acceptor is increased [47]. In general, the following conditions are satisfied in order to have FRET to take place. First, the emission spectrum of donor overlaps with the absorption spectrum of acceptor. Secondly, the distance of the donor and acceptor is in the range 1–10 nm. FRET efficiency (E) can be calculated between S, O-CNQDs and MnO2 using Eq. (1):

$$ E = 1 - \frac{{\tau_{D - A} }}{{\tau_{A} }} = \frac{{R_{0}^{6} }}{{R^{4} R_{0}^{6} }} $$
(1)

τD−A and τA are before and after the fluorescence lifetimes on the combination of energy donor and receptor, respectively. R represents the distance between energy donor and receptor. R0 means the critical distance of Förster, which can be calculated by Eq. (2):

$$ R_{0}^{6} = 8.97 \times 10^{23} \cdot \kappa^{2} \cdot n^{ - 4} \cdot QY_{D} \cdot J\left( \lambda \right) $$
(2)

κ2 represents the dipole orientation factor in the range of 0–4 (κ2 = 2/3). n indicates the refractive index of the solvent, QYD shows the fluorescence quantum yield of donor in the absence of receptor.

In order to clarify the fluorescence quenching mechanism of S, O-CNQDs induced by MnO2, the UV–visible absorption spectrum of MnO2 nanosheets as the acceptor and the fluorescence emission spectrum of S, O-CNQDs as the donor were investigated. Figure 6 shows the UV–visible absorption spectrum of MnO2 nanosheets which largely overlaps with the fluorescence emission spectrum of S, O-CNQDs, indicating that FRET may take place between MnO2 nanosheets and S,O-CNQDs.

Figure 6
figure 6

UV–visible absorption spectrum of MnO2 nanosheets (black) and the fluorescence emission spectrum of S, O-CNQDs (blue)

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The time-resolved fluorescence spectra of S, O-CNQDs (3.0 mg/mL), MnO2-S, O-CNQDs (3.0 mg/mL) and MnO2-S, O-CNQDs (3.0 mg/mL) with GSH (180 μM) were determined and are shown in Fig. 7.

Figure 7
figure 7

The time-resolved fluorescence spectra of S, O-CNQDs (3.0 mg/mL), MnO2-S,O-CNQDs (3.0 mg/mL) and MnO2-S,O-CNQDs (3.0 mg/mL) with GSH (180 μM)

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The fluorescence lifetimes of S, O-CNQDs and MnO2-S, O-CNQDs nanocomposite are determined to be 2.5 and 1.6 ns, respectively. The distance (R) between the MnO2 nanosheets and S, O-CNQDs is 3.83 nm, and transfer efficiency (E) of FRET is 0.36 which were calculated according to Eqs. (1) and  (2). These results indicate that S, O-CNQDS is the donor, while MnO2 is the receptor. Thus, the fluorescence quenching mechanism of MnO2-induced S, O-CNQDs is mainly governed by FRET.

In this work, S, O-CNQDs were deposited on the surface of MnO2 nanosheets to form the non-fluorescence MnO2-S,O-CNQDs nanocomposite owing to FRET. However, in the presence of GSH, MnO2 nanosheets are reduced into Mn2+ and GSH is rapidly oxidized into its oxidized form (GSSG) according to Eq. (3) [48]. Meanwhile, S, O-CNQDs are released and its fluorescence is restored.

$$ {\text{2GSH}} + {\text{MnO}}_{{2}} + {\text{2H}}^{ + } \to {\text{GS}} - {\text{SG}} + {\text{Mn}}^{{{2} + }} + {\text{2H}}_{{2}} {\text{O}} $$
(3)

Determination of GSH in real samples

In order to evaluate the practicability of the fabricated MnO2-S, O-CNQDs nanocomposite, it was applied to detect GSH in real samples. First, different concentrations of GSH were spiked into the diluted biological samples and the GSH injections, respectively. Table 2 summarizes the determination and recovery of GSH in human serum, urine and reduced glutathione injection samples. The recoveries are in the range 97.0–102.7% for biological samples. The results indicate that MnO2-S, O-CNQDs nanocomposite could be a promise fluorescence probe to detect GSH in real samples.

Table 2 Determination of GSH in human serum, urine samples and reduced glutathione injection
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Cytotoxicity assay and fluorescence imaging in living cells

Cytotoxicity assay should be taken into consideration before applying the nanoprobe for imaging of living cells. The cytotoxicity of MnO2-S, O-CNQDs nanocomposite was tested through a standard MTT method. When the concentration of nanocomposite reaches 300 μg/mL, HepG2 cells still keep over 88% viability as depicted in Fig. 8. The results reveal that our nanocomposite has low cytotoxicity and good biocompatibility. Owing to the excellent properties of the nanocomposite, it was applied to monitor the intracellular GSH level in conjunction with a laser scanning confocal microscope.

Figure 8
figure 8

Cell viability of HepG2 cells incubated with MnO2-S, O-CNQDs nanocomposite (0.0–300 μg/mL) for 12 h (n = 3)

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Figure 9 displays the blue fluorescence (λex = 405 nm) and green fluorescence (λex = 488 nm) of the HepG2 cells which have been incubated with 200 μg/mL MnO2-S, O-CNQDs nanocomposite.

Figure 9
figure 9

Laser scanning confocal fluorescence images of HepG2 cells of the experimental group which were incubated with 200 μg/mL MnO2-S, O-CNQDs in bright field (a and d), bright field (b: 405 nm and e: 488 nm) and merge (c and f). Laser scanning confocal fluorescence images of HepG2 cells of the control group which were incubated with 500 mM NEM and 200 μg/mL MnO2-S,O-CNQDs in bright field (g and j), bright field (h: 405 nm and k: 488 nm) and merge (i and l)

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When HepG2 cells are treated with 500 mM NEM as the control group, almost no fluorescence is observed under the laser scanning confocal microscope. These results demonstrate that the nanocomposite with good fluorescence properties can be applied as a “turn-on” probe for bioimaging of GSH in living cells.

Conclusion

In the work, the fluorescence of S, O-CNQDs ( an energy donor) was efficiently quenched by MnO2 (an energy acceptor) based on the FRET effect. The interaction between GSH and MnO2 has a restoring effect on the fluorescence intensity of S, O-CNQDs. As such, a new fluorescence “turn-on” probe for detecting GSH using MnO2-S,O-CNQDs nanocomposite has been developed. The probe displays a good detection capability for GSH in aqueous solution with a wide working range 10–270 μM and a detection limit of 0.307 μM. Our proposed nanoprobe can be applied to real samples (serum, urine and GSH injection solution) for GSH detection with satisfactory results, In addition, the MnO2-S, O-CNQDs nanocomposite can be used for intracellular imaging of GSH. All these results suggest that MnO2-S, O-CNQDs nanocomposite is a promising material which can be utilized for determining GSH in biological, injection samples and human cells. It is anticipated that researchers will continue to explore more novel nanomaterials as the fluorescence probes for effective, low-cost and high-sensitive determination of different analytes.