Introduction

With the development of nanotechnology, nanomaterial-based enzyme mimics (nanozyme) are widely used as alternatives to natural enzymes because of their attractive merits of outstanding tolerance in harsh environments, easy large-scale production, and low cost. The reported enzyme-like activities include the peroxidase-like activity [23], superoxide dismutase-like activity [7], catalase-like activity [17], and so on. The unique characteristics of nanomaterials and the synergistic effect of enzyme-like activities, making nanozymes potential applications in wide fields including biosensing [34], food industry, pharmaceutical processes, and environmental treatment [36]. Since the first research that Fe3O4 nanoparticles possess an intrinsic peroxidase activity [8], an emerging class of nanomaterials such as transition metal oxides/chalcogenides [5, 28, 35], noble metal [3, 22, 32], carbon-based materials [37], metal–organic framework (MOF) [20], and others [16, 25] have been explored as nanozymes.

Recently, two-dimensional (2D) layered materials are attracting in sensing fields due to their unique physicochemical properties originating from their chemical structure [12]. Among them, molybdenum disulfide (MoS2), one of typical 2D materials, has drawn remarkable attentions due to its tunable energy bandgap, intrinsic catalytic activity, durable stability, and natural richness [1]. Each plane is composed by S-Mo-S sandwich-layered structure with weak van der Waals force between layers, which endows MoS2 with graphene-like characteristics, such as unique electronic, high surface area, optical, and chemical properties [19, 33]. Lots of studies reported that the coordinatively unsaturated S/Mo atoms located at the edges of MoS2 nanosheets are active for many catalytic reactions, while the S atoms in-plane domains are usually inserted [10]. However, with the inherent tendency of anisotropic bonding and surface energy minimization, the edges are regular scarce domains of layered materials [29]. Numerous efforts have been made to improve the intrinsic activity of MoS2, including elemental doping, conversion from 2H to 1T, compounding with other materials, etc. [4, 27, 31]. In nanozyme field, the most used improvement of MoS2 is hybridization with other nanomaterials. Cai’s group has designed Pt74Ag26 bimetallic nanoparticle-decorated MoS2 nanosheets for colorimetric determination of H2O2 and glucose [2]. Ma’s group has prepared MoS2-Au composite with peroxidase-like activity for highly sensitive colorimetric detection of Hg2+ [21]. Although these composites demonstrated significant enhancement on enzyme-like catalytic activity compared to pure MoS2, their preparation processes are complicated. Single metal atom doping is considered to be a facile and effective technique to enhance the catalytic activity of MoS2 by allowing the insertion of the original S atom near the doped metal atoms. In natural enzymes, transition metal ions are wrapped around protein substrates as part of metal macrocyclic complexes and exhibit ultra-high catalytic activity and selectivity in biological systems. During enzyme catalysis, the peptide and residue functional groups surrounding the enzyme control the electron density of the active site to an appropriate value matching the energy level of the substrate (reacting species), enabling the transfer of electrons from substrate to product [26]. Li’s group reported that Al-doped MoS2 provided an enhanced catalytic activity for CO oxidation, in which the Al atom doping obviously reduced the band structure and enhanced the conductivity [18]. Shi et al. tuned the energy level of MoS2 via doping with Zn atom, which could accelerate the hydrogen evolution reaction and decrease onset potential [26]. Raza et al. reported that Co-doped MoS2 exhibited improved efficiency for methylene blue degradation [24]. Lei et al. designed an ultrasensitive dopamine sensor based on Mn-doped MoS2 [13]. As far as we know, there was no study explored to investigate the enzyme-like activity of transition metal-doped MoS2.

Hydrogen peroxide (H2O2) is not only a by-product of several highly selective enzymes, but also can be considered as a biomarker for cancer, Alzheimer’s and Parkinson’s due to its damage to tissues and cells [6, 11, 15]. Its rapid and precise detection is therefore a fundamental issue. To date, colorimetric technique for H2O2 detection has particularly attracted wide attentions for point-of-care applications attributed to their great advantages involving simplicity, sensitivity, and low cost [30]. Therefore, in this work, we studied the colorimetric sensing of H2O2 using Co-doped MoS2. Initially, one-step in situ hydrothermal method was utilized for preparation of a serial transition metal-doped MoS2. Interestingly and expectantly, the results showed that the Co-doped MoS2 exhibited superior enzyme-like catalytic activity toward H2O2. The main reason for the highest peroxidase-like activity of Co-doped MoS2 among various transition metal-doped MoS2 including Mn, Ni, Cu, and Fe was revealed by density functional theory (DFT) as well as steady-state kinetic analysis. In addition, determination method of H2O2 catalyzed by Co-doped MoS2 as mimetic peroxidase was then established based on colorimetry. In situ measurement of H2O2 generated from SiHa cells was also fulfilled, expanding the application of Co-doped MoS2 nanosheets in biosensing fields.

Experimental section

Regents and apparatus

L-Cysteine, Co(NO3)2·6H2O, FeSO4·7H2O, CuSO4·5H2O, NiCl2·6H2O, Mn(NO3)2·4H2O, sodium acetate (NaAc), acetic acid (HAc, 99.5%), and dihydroethidium (HE) were purchased from Adamas Reagent Co. Ltd. (Shanghai, China). p-benzoquinone (BQ), isopropanol alcohol (IPA), and terephthalic acid (TA) were supplied by Titan Scientific Co., Ltd. (Shanghai, China; www.tansoole.com) Sodium molybdenum oxide (Na2MoO4) was obtained from Alfa Aesar (Shanghai, China). 30% hydrogen peroxide (H2O2) was supplied by Sigma-Aldrich (USA). 3,3′,5,5′-Tetramethylbenzidine (TMB, 99%), phosphate-buffered saline (PBS, 0.01 M, pH 7.0), fetal bovine serum (FBS), and phorbol 12-myristate 13-acetate (PMA) were bought from Sangon Biotechnology Co., Ltd. (Shanghai, China). The H2O2 assay kit was obtained from Jiancheng Bioengineering Institute (Nanjing, China). Other regents such as glucose, urea, and uric acid were of at least analytical grade and used as-received. The pH value of HAc-NaAc buffer solution (0.1 M) was adjusted by HAc or NaOH (5 M). TMB solution (10 mM) was freshly prepared by dissolving TMB in anhydrous ethanol. Ultrapure water (18.2 MΩ cm) used in the whole experiments was obtained by Millipore ultrapure system.

Surface morphologies of the prepared MoS2 were identified by transmission electron microscopy (TEM, FEI spirit T12, USA) by drying 10 μL sample solution on Cu-grids with carbon film. Field emission scanning electron microscopy (FE-SEM) images were obtained on ITO-glass using Hitachi S-4800 (Japan). X-ray diffraction (XRD) was operated on an UltimaIV X-ray Cu Ka radiation diffractometer for element analysis by drying sample solution on ITO-glass. X-ray photoelectron spectroscopy (XPS) measurement, which was used for elemental analysis, was performed on Thermo ESCALAB 250XI. All colorimetric measurements were performed on UV-2600 ultraviolet–visible (UV–Vis) spectrometer (Shimadzu, Kyoto, Japan). Steady-state kinetic assays and fluorescence spectra were recorded by Synergy™ HTX Multi-Mode microplate reader (Biotek Instruments, USA).

Materials synthesis

MoS2-based materials were synthesized according to a modified one-pot hydrothermal route reported by Guo et al. [9]. For preparation of pure MoS2, an aqueous solution of 2.0 mM Na2MoO4 and 4.0 mM L-cysteine was stirred at room temperature for 15 min and then transferred into a 100 mL Teflon-lined stainless-steel autoclave. The autoclave was heated at 200 °C for 12 h and cooled down to room temperature naturally. Subsequently, the as-obtained product was collected by centrifugation with a speed of 12,000 rpm for 20 min. The precipitate was washed with DI water for three times and re-dispersed in 50 mL DI water. Besides, Co-, Fe-, Cu-, Ni-, and Mn-doped MoS2 nanomaterials were synthesized using the same method as above in the presence of 0.15 mM of the corresponding metal ion (Co(NO3)2, FeSO4, CuSO4, NiCl2, and Mn(NO3)2) in a mixed solution of Na2MoO4 and L-cysteine, respectively.

The peroxidase-like activity of Co-doped MoS2

The spectroscopic determination of H2O2 by Co-doped MoS2 was investigated as follows: 1.375 mL NaAc (0.1 M, pH 4.0) buffer was mixed with 50 μL Co-MoS2 (0.325 mg/mL) and 10 μL H2O2 (1 mM), and then, 75 μL TMB (0.5 mM) was added immediately and mixed with vortex mixer. After reaction for 10 min, the UV–Vis absorption spectra were recorded at 652 nm. The concentration of Co-doped MoS2 was optimized by similar procedure except that the dosage of Co-doped MoS2 (0–100 μL) was changed. The influence of pH and temperature on catalytic activity of Co-doped MoS2 was also inquired by the above-mentioned method, expect that the pH value (3.0–10.0) and temperatures (4–50 °C) of the reaction solution were varied, respectively. For each determination, three independent measurements were conducted.

Steady-state kinetic assays

Steady-state kinetic measurements of the catalytic reactions were conducted at room temperature in 96-well plates with 200 μL reaction solution (0.1 M NaAc, pH 4.0) with 7.1 μL Co-doped MoS2 (0.325 mg/mL) in the presence of 10 μL TMB (10 mM) and different concentrations of H2O2 (5, 10, 20, 30, 40, 50, 75, 125, 250, 375 mM), while for TMB as the substrate, the kinetic assay was performed by a similar method with diverse TMB concentrations (0.1–1 mM) in the presence of 10 mM H2O2. All reactions were performed by measuring the absorbance at 652 nm (A652nm) at various reaction times. The obtained A652nm were back-converted to oxTMB concentration by Beer–Lambert law, A = εbC, where the molar absorption coefficient ε was 39,000 M−1 cm−1, and path length of vitric cuvette b was 0.625 cm. Kinetic parameters were calculated according to the Michaelis–Menten equation: 1/v = Km/Vm[S] + 1/Vm, in which ν is the initial velocity, which can be calculated by the initial slope of A652nm changes with time, Vmax is the maximum velocity, [S] is the concentration of substrate (H2O2 or TMB), and Km is the Michaelis–Menten constant.

Performance of Co-doped MoS2 on H2O2 determination

For quantitative analysis of H2O2, the colorimetric assay was performed at 25 °C in a 1.5 mL reaction buffer solution (0.1 M acetate buffer, pH 4.0) containing 50 μL Co-doped MoS2 (0.325 mg/mL) as catalyst in the presence of different concentrations of H2O2, and then, 75 μL TMB (0.5 mM) was added and mixed immediately. After 10 min incubation, the spectrophotometric measurement was recorded at 652 nm. Three independent measurements were performed for each H2O2 concentration.

Real sample detection

SiHa cells were purchased from the Chinese Academy of Sciences Cell Bank. They were cultured in Dulbecco's modified eagle medium (supplemented with 10% FBS, and 1% penicillin/streptomycin) and maintained in an atmosphere of 5% CO2 at 37 °C. In order to assess the release of H2O2 from SiHa cells, the cells (4 × 104 cells/plate) were dropped into 96-well microplate for 12 h and then washed three times with PBS solution. After that, 20 μL of PMA solution (75 ng/mL) and 80 μL of PBS were added successively and incubated for 30 min. Furthermore, Co-doped MoS2 (0.325 mg/mL, 10 μL), TMB (50 μL, 0.5 mM), and acetate buffer (100 μL, pH 4.0) were added subsequently for incubation 10 min. Finally, the absorbances at 652 nm were recorded by a microplate reader.

Results and discussion

Synthesis and characterization of Co-doped MoS2

The Co-doped MoS2 materials were prepared by a one-pot hydrothermal method using Na2MoO4 and Co(NO3)2 as precursors and L-cysteine as a reductant (Fig. 1a). The scanning electron microscopy (SEM) images (Fig. 1b) display Co-doped MoS2 consisting of a fairly homogeneous two-dimensional nanosheets structure with high surface area and volume-to-surface ratio. Similar structures also appear in MoS2 and other transition metals (Ni, Fe, Cu, Mn)-doped MoS2 (Fig. S1A–E). An ultrathin lamellar structure with ample graphene-like wrinkles and folds is observed in transmission electron microscopy (TEM) image (Fig. 1c), further implying the typical two-dimensional nanosheets structure of Co-doped MoS2.

Figure 1
figure 1

a Schematic description of the Co-doped MoS2 synthesis process. b SEM and c TEM images of Co-doped MoS2. The high-resolution XPS spectra of d Mo3d and e S2p. a–f stands for pure MoS2, Mn-doped MoS2, Fe-doped MoS2, Ni-doped MoS2, Cu-doped MoS2, and Co-doped MoS2, respectively

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The typical X-ray diffraction (XRD) patterns of MoS2 and Co-doped MoS2 are shown in Fig. S2. A diffraction peak at 14.4° can be well indexed to (002) of MoS2 (JCPDS card no. 37–1492) and Co-doped MoS2 [14, 19]. There is no other crystal phase observed after Co doping except the intensity of (002) increased, indicating the Co doping can enhance the crystal phase of MoS2. The composition of obtained Co-doped MoS2 was further verified by X-ray photoelectron spectroscopy (XPS). As shown in Fig. S3, combined with the overall XPS spectrum and the high-resolution XPS spectra of each doping element, the successful doping of Co and other transition metals into MoS2 was demonstrated. The content of Co incorporation in Co-doped MoS2 is calculated as 1.3 atom %. It is noteworthy that the characteristic signal of O 1s is detected probably from the adsorbed O2 on the surface of MoS2. Besides, from the high-resolution spectra of Mo 3d (Fig. 1d) and S 2p (Fig. 1e), the binding energy of Co-doped MoS2 shows a huge shift compared with that of MoS2. In Fig. 1d, Co-doped MoS2 displays two main peaks at 227.7 and 230.8 eV, originating from the doublet of Mo 3d5/2 and Mo 3d3/2 (curve f). It is clear that these two peaks are negatively shifted by 0.4 eV when compared to pure MoS2. In Fig. 1e, the binding energies of S 2p3/2 and S 2p1/2 are negatively shifted by 0.5 eV after Co incorporation. The similar phenomenon is also discovered in Ni-doped MoS2 (curve d) and Cu-doped MoS2 (curve e), indicating the increased electronic density in MoS2 after these elements’ doping [26].

Mimic enzymatic activity investigation

To study the enzyme-like activity of Co-doped MoS2 nanosheets, catalytic reaction was performed in the presence of H2O2 with TMB as substrate. As displayed in Fig. 2a, with the coexistence of TMB, H2O2, and Co-MoS2 (curve a), a remarkable color change reaction occurs and a strong absorbance appears at 652 nm, whereas the control experiments show negligible absorbance in the absence of Co-doped MoS2 or H2O2 (curve b, c, d). This result indicates the peroxidase-like activity of Co-doped MoS2 nanosheets, which can catalyze the oxidation of colorless TMB to blue oxTMB with the help of H2O2. Furthermore, the peroxidase-like activities of MoS2 doped with various transition metal atoms were studied (Fig. 2b). Compared with pure MoS2, the catalytic activities of Fe-doped MoS2, Cu-doped MoS2, and Co-doped MoS2 toward H2O2 are enhanced significantly, and Co-doped MoS2 material possesses the highest activity. In contrast, Ni-doped MoS2 and Mn-doped MoS2 show suppressive absorbance at 652 nm. To reveal the enhancement in the peroxidase-like activity of Co-doped MoS2, the density of states (DOS) was calculated by DFT and a (3 × 3 × 1) Monkhorst–Pack mesh was used for the Brillouin-zone integrations to be sampled (Fig. S4). As shown in Fig. 3, the DOS of transition metal-doped MoS2 around the Fermi level increases in varying degrees compared to MoS2, suggesting the doping of transition metal increases the metallicity of MoS2, which in turn leads to easier electron escape. The distribution of projected density of states (PDOS) further proves that the doped metal can act as active sites for the interaction with the substrate. The d-band center is generally used to indicate the ability of a substance to adsorb and desorb reactants. The farther the d-band center is from the Fermi level, the more favorable the desorption of reactants, while the opposite is true for the adsorption of reactants. Herein, the d-band centers of Ni-doped MoS2 and Co-doped MoS2 are − 0.697 eV and − 0.728 eV, respectively. Combined with their higher density of states at the Fermi energy level, it is assumed that they should have higher catalytic activity. These results of DFT are consistent with that of XPS analysis.

Figure 2
figure 2

a UV–Vis spectra of HAc-NaAc buffer solution (0.1 M, pH 4.0) containing a 1 mM H2O2 + 0.5 mM TMB + Co-doped MoS2, b 1 mM H2O2 + 0.5 mM TMB, c 0.5 mM TMB + Co-doped MoS2, d 0.5 mM TMB, respectively. b The effect of doping element on peroxidase-like activity. c In the coexistence system of 1 mM H2O2, 0.5 mM TMB, 0.325 mg/mL Co-doped MoS2, and 0.1 M HAc-NaAc buffer (pH 4.0), the relative activity changed after adding 10 mM IPA or BQ. d Schematic description of Co-MoS2 nanosheets exerting peroxidase-like activity

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Figure 3
figure 3

The DOS and d-band center of a MoS2 (001), b Co-doped MoS2 (001), c Cu-doped MoS2 (001), d Fe-doped MoS2 (001), e Mn-doped MoS2 (001), and f Ni-doped MoS2 (001) by DFT

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In general, reactive oxygen species like hydroxyl radical (•OH) and superoxide radical (•O2−) could be easily generated from H2O2 in nanozyme catalytic reaction. Therefore, to understand the possible catalytic mechanism of peroxidase-like activity, different scavengers (IPA as •OH scavenger and BQ as •O2− scavenger) were added to the reaction system. As shown in Fig. 2c, it is obvious that the significant decrease in the relative activity of Co-doped MoS2 nanosheets comes alongside the addition of BQ, while for IPA, the change is weak. This result suggests that •O2− plays a major role in the catalytic system. Additionally, TA and HE were employed as fluorogenic indicators for •OH and •O2− to identify the type of radicals produced in the catalytic reaction. When Co-doped MoS2 nanosheets and H2O2 co-existed, a strong fluorescent signal was observed at 610 nm with the addition of HE (Fig. S5A), while little change in fluorescent signal was found at 380 nm after adding TA (Fig. S5B). This result further reveals that the catalytic behavior of Co-doped MoS2 is due to the production of •O2− and •OH, and the former exerts a greater influence on the activity of peroxidase-like enzymes. The catalytic process of Co-doped MoS2 nanosheets is demonstrated in Fig. 2d.

Optimization of assay condition

In order to demonstrate the best performance of this colorimetric assay, the preparation and reaction conditions were optimized. Firstly, the effect of doping amount on the catalytic activity of Co-doped MoS2 was investigated (Fig. 4a). The concentrations of Co2+ in precursor are varying from 0 to 0.25 mM. From Fig. 4a, the value of A652nm increases obviously from 0 to 0.15 mM and then slightly decreases with the Co amount continuously increasing, which may be attributed to the fact that excessive doping amount cannot be fully incorporated into MoS2. Therefore, Co doping amount was set as 0.15 mM. As is known to all, dosage of the catalyst is crucial for catalytic reaction, and the influence of different concentrations of Co-doped MoS2 was studied (Fig. 4b). The absorbance intensity of reaction solution increases gradually with the increase in Co-doped MoS2 concentration. Nevertheless, when the amount of Co-doped MoS2 exceeds 50 μL, the large concentration of Co-doped MoS2 tends to gather and form black flocculent precipitates under acidic conditions, resulting in an unstable absorbance of the system during the detection process, which in practice shows that the absorbance is suddenly low and high. So, 50 μL was chosen as the optimal concentration of Co-doped MoS2 nanosheets. As we know, natural enzyme (such as HRP) usually suffers from instability and inactivation under extreme conditions. In contrast, the superiority of nanozyme over natural enzyme is the tolerance and robustness under extreme determination conditions. The catalytic activity of Co-doped MoS2 nanosheets was investigated with diverse pH values (3.0–10.0) at various temperatures (4, 25, and 50 °C). As can be seen in Fig. 4c, the peroxidase-like activity of Co-doped MoS2 is much higher in acidic media (pH 3.0–5.0) than in neutral and basic conditions at all temperatures. The optimal activity is observed at pH 4.0, which is similar to that of HRP. In the case of temperature, the effect is much smaller than that of pH value, in which superior catalytic activity over a wide range of temperature even under high (50 °C) or low (4 °C) temperature conditions was shown. Thus, pH value of 4.0 and temperature of 25 °C are adopted as standard conditions for the following colorimetric analysis. Considering the above results, endurance and robustness of Co-doped MoS2 as peroxidase mimetics under extreme environments are excellent.

Figure 4
figure 4

The effect of a content of Co, b dosage of Co-doped MoS2, and c pH and temperature on peroxidase-like activity

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Steady-state kinetic assays of Co-doped MoS2 nanosheets

To better understand the peroxidase-like catalytic activity of Co-doped MoS2 nanosheets, it is necessary to perform apparent steady-state kinetic analysis to acquire the kinetic parameters such as Michaelis–Menten constant (Km) and maximum initial velocity of the reaction (Vmax). As shown in Fig. S6, typical Michaelis–Menten curves are obtained by varying concentrations of TMB or H2O2 under similar conditions. A well-defined linear fit is found from the corresponding Lineweaver–Burk plots (Fig. S6B, D), and Km and Vmax are calculated according to the Michaelis–Menten equation. The corresponding parameters were summarized and compared with natural horseradish peroxidase (HRP) (Table 1). It is well known that Km represents the enzyme affinity of nanozyme to substrate. Low Km value indicates high affinity to substrate. Co-doped MoS2 demonstrates a higher affinity for TMB as well as a larger Vmax value for H2O2 as substrate than HRP. In addition, the Km for H2O2 is much higher than that for TMB, suggesting that in high concentrations of H2O2, the Co-doped MoS2 nanosheets can react with relatively low amount of substrate to achieve maximum catalytic activity.

Table 1 Comparison of Km and Vmax between Co-doped MoS2 and HRP
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Besides, the Km and Vmax values for each MoS2 material are summarized in Table S1. From Table S1, Co-doped MoS2 nanosheets have the lowest Km and largest Vmax, suggesting Co-doped MoS2 owns the best affinity for H2O2 and rapidity of the catalytic reaction. However, the Vmax of Ni-doped MoS2 is only 0.01 μM s−1, which is 7 times lower than even MoS2. Given that the Co-doped MoS2 has a better performance in terms of both electron density, active site, adsorption and desorption capacity, affinity, and reaction rate, it has the highest peroxidase-like activity.

Colorimetric detection H2O2 using Co-MoS2 nanosheets

Based on intrinsic peroxidase-like activity of Co-doped MoS2 nanosheets, determination of H2O2 was conducted using the chromogenic reaction catalyzed by Co-doped MoS2 in the presence of TMB. Figure 5a, b shows the absorption spectra of TMB increase with the concentration of H2O2 increasing, and the absorbance at 652 nm is linearly dependent on the concentration of H2O2 from 0.0005 to 25 mM. Interestingly, two linear detection ranges are obtained. It is found that A652nm shows a good linear relationship to H2O2 concentration in a range of 0.0005–1 mM with regression coefficient (R2) of 0.9989 (Fig. 5c), while in the range of 1–25 mM, as shown in Fig. 5d, the A652nm is proportional to the logarithmic concentration of H2O2 (R2 = 0.9944). The limit of detection is calculated to be 0.3 μM (S/N = 3). These results suggest that this colorimetric assay could be applied to measure the amount of H2O2 in a broad range, which is superior to others reported in the literature, as listed in Table S2.

Figure 5
figure 5

a UV–Vis spectra of Co-doped MoS2 measuring various concentrations of H2O2 with pH of 4.0, TMB concentration of 0.5 mM, and reaction time of 10 min. b The dependency of the absorbance at 652 nm on the H2O2 concentration from 0.5 μM to 25 mM. c and d represent the calibration curves in the range of 0.5 μM to 1 mM and 1 mM to 25 mM, respectively

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The specificity was examined by using present assay to catalyze glucose, urea, uric acid, ascorbic acid, Cu2+, Mn2+, Ni2+, PO43−, respectively. Compared with H2O2, negligible interference is discovered for interferent species although their concentrations are 10 times higher than that of H2O2 (Fig. 6a). In the long-term stability test, Co-doped MoS2 suspension was sealed with Parafilm and stored at room temperature. Before use, the suspension was ultrasonicated for 10 min. There was no obvious decrement in A652nm for 1 mM H2O2, and 89.3% of the initial signal was retained after 30 days storage (Fig. 6b). Such observations imply that our catalytic system possesses feasibility for H2O2 detection. The repeatability was investigated by detection 1 mM H2O2 under optimal conditions for ten individual measurements, which yielded RSD of 2.67% (Fig. S7A). For reproducibility, five independent suspensions containing Co-doped MoS2 nanosheets were synthesized under same conditions, and the RSD generated from the A652nm for 1 mM H2O2 was evaluated to be 4.18% (Fig. S7B). These results verify that the Co-doped MoS2 nanosheets have decent repeatability and reproducibility.

Figure 6
figure 6

a Normalized absorbance at 652 nm detected by Co-doped MoS2 in the presence of 1 mM H2O2 and other interferent species. b Response obtained from Co-doped MoS2 measuring 1 mM H2O2 after storage of different periods. c Comparative analysis of the level of H2O2 in SiHa cells induced by different concentrations of PMA between our proposed method and an H2O2 kit. Error bars represent the standard deviations from three parallel measurements

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Determination of H2O2 in SiHa cells

H2O2 is a biomarker of cancer, which is highly expressed in cancer cells. Therefore, it is necessary to quantitatively determine H2O2 and monitor the release of H2O2 in cells. In our study, in situ detection of H2O2 released from SiHa cells was performed using PMA as a stimulant. As shown in Fig. 6c, 75 ng/mL PMA has the strongest induction of H2O2. In comparison with the commercial H2O2 kit, the detection results show no significance between the two assays with a confidence interval of 95% (p > 0.05). These results indicate that Co-doped MoS2 is feasible and reliable to detect H2O2 in SiHa cells.

Conclusions

To sum up, we have successfully synthesized a serial transition metal-doped MoS2 by a facile in situ hydrothermal method and their enzyme-like activities were investigated against TMB as chromogenic substrate in the presence of H2O2. When the doping element was cobalt (Co), the highest peroxidase-like activity was obtained, which was much better than pure MoS2 and other doped MoS2. Furthermore, DFT and steady-state kinetic analysis revealed the main reason for the highest peroxidase-like activity of Co-MoS2, which is due to its superior performance in electron density, active sites, adsorption and desorption capacity, affinity, and reaction rate. •O2− was also confirmed to play a major role in peroxidase-mimicking reactions. Meanwhile, Co-doped MoS2 exhibited long-term stability and excellent endurance and robustness under extreme environments. Two linear ranges of 0.0005–1 mM and 1–25 mM were observed for H2O2 by this colorimetric assay. The detection limit is as low as 0.3 μM. Besides, in situ measurement of H2O2 generated from SiHa cells was fulfilled, fully confirming the great practicability of the proposed method in biosensing fields. Our approach can also be potentially extended to design a series of other elements (such as Pt, Pd, B, N, and C)-doped MoS2 as nanozymes. This work provides an alternative strategy for designing highly sensitive colorimetric sensors for biological and clinical applications. However, the poor selectivity limits the practical application of nanozyme in complicated environments. Thus, further studies such as cascading with natural enzyme and surface functionalization are progressed to circumvent the above issue.