1 Introduction

Glucose is the energy source of human living cells and an important substance in the metabolic process, but it is easily absorbed by the blood, leading to excessive blood sugar, causing metabolic disorders, vascular disease, organ disease and other serious diseases that endanger human health [1, 2]. Therefore, it is important and necessary to establish a simple, accurate and rapid method for detecting glucose. At present, electrochemical sensing, surface-enhanced Raman scattering, colorimetry and fluorescence are often used to monitor glucose content [3,4,5,6]. Among them, the colorimetric method has received widespread attention because of its economy, simplicity, and its ability to be monitored through visual screening. The classic colorimetric method uses glucose oxidase (GOx) to oxidize glucose to gluconic acid and hydrogen peroxide (H2O2), and monitors the glucose content through the color change process between H2O2 and natural peroxidase [7,8,9,10]. However, natural enzymes have limitations such as poor stability, variability, and difficulty in recycling, which are severely restricted in practical applications [11, 12].

Ever since the first artificial nanoenzyme Fe3O4 was reported, many peroxidase mimics such as precious metals, metal oxides, transition metal double halides have been developed [13]. Fe3O4 nanoparticles have peroxidase-like activity and can be used for wastewater detection through color changes [14]. In recent years, heterogeneous nanozymes have attracted widespread attention due to their advantages such as good stability, adjustable catalytic sites, and easy synthesis [15,16,17,18]. Graphene (RGO) has unique properties such as electrical conductivity, thermal stability, chemical stability, and π−π conjugated structure system, and exhibits a peroxidase activity that can catalyze the formation of active hydroxyl radicals from H2O2 [19, 20]. It is used as a matrix material for heterogeneous mimic enzymes to enhance the sensitivity of the colorimetric sensor in the detection process [21, 22]. For instance, anoparticles such as ZnO, Co3O4, CuO are often loaded on the surface of graphene for glucose sensing and detection [23]. However, the currently reported graphene-based nanozymes have the disadvantages of small specific surface area, less exposed active sites, and slightly slower colorimetric detection signals, so that their peroxidase activity needs to be further improved [24].

In recent years, the Metal organic frameworks (MOFs) as a class of porous materials has attracted much more attention due to their unique characters such as large porosity, large specific surface area and adjustable pore size [25, 26]. Due to their large specific surface area, adjustable pore size, and strong stability, they are used in adsorption, separation, biosensing and catalysis [27, 28]. The metal centers and organic ligands in the MOFs structure can be used as transport carriers for colorimetric sensing; its ordered microporous framework can provide abundant adsorption and catalytic sites [29,30,31], which can be used to monitor glucose in real samples [32]. Among them, MOFs with transition metal Fe3+ as the active center can improve the peroxidase ability of graphene and play a synergistic effect in the activation of H2O2 [33]. It also has similar chemical properties and excellent performance to mimic enzymes, making it an ideal candidate for enhancing the activity of graphene-based heterogeneous mimic enzymes [34]. Therefore, using MOFs (Fe) to modify graphene-based heterogeneous nanozymes is an effective method to further improve its peroxidase activity.

In this work, we proposed a facile one-step route to prepared NH2-MIL-88B(Fe) and Fe3O4 modified reduced graphene oxide (RGO) mimic enzymes (NH2-MIL-88B(Fe)@MRGO), where NH2-MIL-88B (Fe) servers as the catalytically active component. These mimic enzymes combine the peroxidase activity of Fe3O4, graphene, and NH2-MIL-88B(Fe) in one, which provide a huge active center for absorption and catalysis, leading to an enhanced peroxidase activity for detecting glucose. At the same time, a glucose detection system based on the cascade reaction of glucose oxidase (GOx) and enhanced heterogeneous nanozyme (NH2-MIL-88B(Fe)@MRGO) was constructed. It provides an effective strategy for the detection of H2O2 and glucose with high sensitivity, good stability and simple colorimetric sensing, and has great application prospects in biological analysis, disease diagnosis and pollution treatment.

2 Experimental

2.1 Chemicals and Reagents

Ferric chloride hexahydrate (FeCl3·6H2O), Ethylene glycol (EG), sodium acetate (NaAc), acetic acid, hydrogen peroxide(30% wt%, H2O2) were obtained from Tianjin Sailboat Chemical Reagent Co. Ltd. Graphite, dimethyl aminoterephthalate (NH2-BDC) were obtained from Alfa Aesar. Glucose oxidase (GOx, 200 Umg−1) was purchased from Sigma-Aldrich and stored in the refrigerator at − 20 °C. 3,3′,5,5′-Tetramethylbenzidine (TMB), glucose, fructose, maltose, lactose were purchased from Aladdin. All the reagents above were of analytical reagent grade and used without further purification. Deionized water was used throughout the experiment.

2.2 Preparation of GO and GO-COOH

Graphene oxide (GO) was prepared from purified natural graphite by a modified Hummers method [35]. GO-COOH was synthesized as follows: 5 g NaOH were dissolved in 100 mL of deionized water. After the solid was cooled down to room temperature, 100 mg GO and 5 g sodium chloroacetate was added into the above solution and sonicated for 30 min. Finally, the products were thoroughly washed several times with ethanol and water until the pH of the product reached about 7.0, and dried in vacuum overnight.

2.3 Preparation of MRGO and MRGO-COOH

Magnetic oxidized graphene (MRGO) was synthesized by a hydrothermal method as precious describe [36]. Briefly, 100 mg GO was dispersed into a solution consists of 60 mL EG. The mixture was ultrasonically treated for 10 min before 1.620 g FeCl3, 0.353 g sodium citrate, and 4.320 g NaAc were introduced and stirred for 1 h at room temperature. After that, the mixture was transferred to 30 mL Teflon-lined stainless autoclaves and heated for 12 h at 200 °C under autogenous pressure. After the autoclave was cooled down to room temperature, the precipitate was filtered, washed with distilled water, and ethanol for several times, and dried in a vacuum oven at 60 °C for 12 h.

100 mg MRGO was dissolved in deionized water firstly and sonicated for 30 min, 5.00 g NaOH and 5.00 g NaClO3 were added into the above solution. The mixture was sonicated for 1 h. The as-prepared product, carboxylated magnetic oxidized graphene (MRGO-COOH), was washed by ethanol until the pH of the product reached about 7.0. Afterward, the product was dried in a vacuum oven at 60 °C for 12 h.

2.4 Preparation of NH2-MIL-88B(Fe)

The synthesis of NH2-MIL-88B(Fe) NPs was based on the previous report [37]. Briefly, 0.675 g FeCl3·6H2O and 0.225 g NH2-BDC were dissolved in 15 mL DMF and sonicated until a homogeneous solution was observed. The mixed solution was ultrasound and then transferred to a Teflon-lined stainless-steel autoclave and was heated at 110 °C for 30 h. After cooling to room temperature, the final product was centrifugated, purified by a double treatment in ethanol and DMF, and dried in a vacuum at 60 °C.

2.5 Preparation of NH2-MIL-88B(Fe)@GO and NH2-MIL-88B(Fe)@MRGO

The process of synthesizing NH2-MIL-88B(Fe)@GO and NH2-MIL-88B(Fe)@MRGO was as follows: 0.675 g FeCl3·6H2O and 0.225 g NH2-BDC were dissolved in 15 mL DMF and sonicated for 30 min. 100 mg GO-COOH or MRGO-COOH was added into the above solution and sonicated until a homogeneous solution was observed. The mixed solution was then transferred to a Teflon-lined stainless-steel autoclave and was heated at 110 °C for 24 h. After cooling to room temperature, the final product was centrifugated, purified by a double treatment in ethanol and DMF, and dried in a vacuum at 60 °C.

2.6 Kinetics Measurements

The peroxidase-like activity of NH2-MIL-88B(Fe)@MRGO was investigated by monitoring the absorbance of TMB at 652 nm with UV–vis spectrophotometer. The stock solution of TMB(1 mM) was prepared with anhydrous ethanol using 0.4 mg mL−1 NH2-MIL-88B(Fe)@MRGO in 1 mL of acetate buffer (0.2 M, pH = 4) in the presence of 50 μL of TMB (1 mM) and H2O2 (20–800 μM) as the substrates with a total reaction volume of 1.15 mL at room temperature. The Michaelis–Menten constant was calculated by using Lineweaver–Burk plots of the double reciprocal of the Michaelis–Menten equation: V = Vmax × [S]/(Km + [S]). where V is the initial velocity, Vmax represents the maximal reaction velocity, [S] corresponds to the substrate concentration, and Km is the Michaelis–Menten constant.

2.7 Detection of H2O2 and Glucose

A series of H2O2 solution with the concentration range from 20 to 800 μM were freshly prepared. The colorimetric assay was constructed as follows: 50 μL of NH2-MIL-88B(Fe)@MRGO particles (0.4 mg·mL-1) were mixed with 1 mL of acetate buffer (0.2 M, pH = 4), 50 μL of TMB(1 mM) stock solution and 50 μL of H2O2 (20–800 μM) solution. After incubation at 45 °C for 20 min, NH2-MIL-88B(Fe)@MRGO particles were magnetically removed from the reaction system. The supernatant was measured by a UV spectrophotometer and the maximal absorbance of oxidized TMB was recorded at 652 nm.

For the determination of Glucose, 20 μL GOx (20 mg mL−1) was added into a phosphate buffer solution (PBS, 0.01 M, pH = 6.0) containing different amounts of glucose and incubated at 37 °C for 20 min. After that, the above solution was added into a 200 μL of acetate buffer (0.2 M, pH 6.0) containing 50 μL of TMB (1 mM) and 300 μL NH2-MIL-88B(Fe)@MRGO (0.4 mg mL−1) in a total volume of 0.875 mL. The resulted solution was incubated at 37 °C for 15 min and the supernatant was subjected to absorbance spectroscopy measurement.

3 Results and Discussion

3.1 Characterization of NH2-MIL-88B(Fe)@MRGO and Its Analogues

The morphology of the as-prepared samples, NH2-MIL-88B(Fe), calcined NH2-MIL-88B(Fe), NH2-MIL-88B(Fe)@GO, NH2-MIL-88B(Fe)@MRGO, was firstly investigated by TEM characterization. Figure 1a shows that NH2-MIL-88B(Fe) has a hexagonal biconical prism structure with a length of ~  700 nm. It can be seen from Fig. 1b that there are nanoparticles on the surface of the calcined NH2-MIL-88B(Fe), but the basic morphological structure of the NH2-MIL-88B(Fe) derivative is maintained. We see that NH2-MIL-88B(Fe) is uniformly distributed on the surface of GO, and the morphology and structure of the two themselves have not changed (inserted TEM image in Fig. 1c), indicating the success of the binary material NH2-MIL-88B(Fe)@GO preparation. After introduction of Fe3O4, we find that Fe3O4 and NH2-MIL-88B(Fe) are adhered well to the surface of wrinkled RGO.

Fig. 1
figure 1

Morphology of a NH2-MIL-88B(Fe), b calcined NH2-MIL-88B(Fe), c NH2-MIL-88B(Fe)@GO, d NH2-MIL-88B(Fe)@MRGO

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The crystallographic structures of the samples were further characterized using XRD technique. As shown in Fig. 2a. All the peaks of the as-prepared sample coincide with the standard NH2-MIL-88B(Fe) [37]. Definitely, the diffraction peaks at 9.03°, 10.11° and 12.94°,16.52°,18.28°,20.61° are assigned to the diffraction of (002), (101), (102), (103), (200), (201) planes of metallic NH2-MIL-88B(Fe), respectively (see curve a). This indicates that NH2-MIL-88B(Fe) is successfully synthesized in this study. Once NH2-MIL-88B(Fe) was calcined at 400 ºC for 10 min, the XRD patterns of calcined samples was in agreement with that of Fe3O4 (see curve b), which demonstrate that NH2-MIL-88B(Fe) has transformed into Fe3O4. Notably, the diffraction peaks of NH2-MIL-88B(Fe) and GO were both observed for NH2-MIL-88B(Fe)@GO and NH2-MIL-88B(Fe)@MRGO, indicating NH2-MIL-88B(Fe) were deposited on the layers of GO and MRGO respectively. In particular, in NH2-MIL-88B(Fe)@MRGO the diffraction peaks of Fe3O4 are seen, indicating Fe3O4 microspheres are successfully introduced combing with the SEM observation of NH2-MIL-88B(Fe)@MRGO (Fig. 1d).

Fig. 2
figure 2

The XRD pattern a FTIR spectra b of NH2-MIL-88B(Fe) (curve a), calcined NH2-MIL-88B(Fe) (curve b), NH2-MIL-88B(Fe)@GO (curve c), NH2-MIL-88B(Fe)@MRGO (curve d)

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Fourier-transform infrared (FTIR) was used to investigate the functional groups on the surface of NH2-MIL-88B(Fe)@MRGO. As shown in Fig. 2b, the characteristic peaks of the as-prepared NH2-MIL-88B(Fe) are almost identical to C=C at around 1584 cm (see curve b) 1. The peaks at 1372 cm−1 are attributed to the symmetric stretching of C–N. These characteristic peaks also appeared in FTIR spectrum of NH2-MIL-88B(Fe)@RGO. This observation indicates MRGO was successfully composited with NH2-MIL-88B(Fe). Besides, the peak at 590 cm−1 belonged to the Fe–O symmetric stretching of Fe3O4 is seen. This observation confirms the successful combination of Fe3O4 with NH2-MIL-88B(Fe)@RGO.

The nitrogen adsorption–desorption isotherms and pore size distribution were conducted for calcined NH2-MIL-88B(Fe), NH2-MIL-88B(Fe)@GO and NH2-MIL-88B(Fe)@MRGO (Fig. S1). The Brunauer–Emmett–Teller(BET) surface areas of calcined NH2-MIL-88B(Fe), NH2-MIL-88B(Fe)@GO and NH2-MIL-88B(Fe)@MRGO were 52.72 m2g−1, 58.97 m2g−1, 113.07 m2g−1, respectively. Thus, the introduction of graphene and Fe3O4 results in the increase in the specific surface. Additionally, they all exhibited a type-IV isotherm being representative of mesoporous structure which was prerequisite for an efficient sensing device.

3.2 Peroxidase Mimic Activity

To investigate the catalytic activity of various mimic enzymes, the typical peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) was chosen as the chromogenic substrate in the presence of H2O2. The catalytic activities of four mimic enzymes (NH2-MIL-88B(Fe) and its analogues) were monitored using UV–vis absorption spectroscopy to quantitatively analyze the amount of decomposed H2O2 (Fig. 3). In the absence of mimic enzymes or H2O2, no oxidation reaction of TMB occurred as no change in solution color was observed, indicating that both mimic enzymes and H2O2 were indispensable for the catalytic reaction [38,39,40]. Once the as-prepared mimic enzymes and H2O2 were introduced, the strong characteristic absorption peak at 652 nm was observed. Meanwhile, a blue color was seen (see insert in Fig. 3), a characteristic chromatogenic reaction for the formation of charge-transfer complexes which originates from the one-electron oxidation of TMB (oxTMB) [41, 42]. These results indicated that these as-prepared mimic enzymes can efficiently catalyze the oxidation of TMB by H2O2 and have a strong intense colorimetric response to H2O2. Consequently, well-performed mimic enzymes based on NH2-MIL-88B(Fe) are successfully constructed as the sensitive colorimetric sensing platform. Of note, the adsorption peaks for NH2-MIL-88B(Fe) and calcined NH2-MIL-88B(Fe) mimicking enzymes is slightly shifted to the left, compared with the NH2-MIL-88B(Fe)@GO and NH2-MIL-88B(Fe)@MRGO. The reason is that the composite mimic enzymes have a π-π conjugated structure system originating from GO and MRGO, which will slightly reduce the ultraviolet absorption energy of the reaction system. Eventually, a slight increase in λmax and a slight red shift of the absorption peak to the right are observed for NH2-MIL-88B(Fe) and calcined NH2-MIL-88B(Fe).

Fig. 3
figure 3

Absorption spectra and digital photos of different colorimetric reaction systems: NH2-MIL-88B(Fe) and calcined NH2-MIL-88B(Fe) (a), NH2-MIL-88B(Fe)@GO (b), NH2-MIL-88B(Fe)@MRGO (c)

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3.3 Optimization of Experimental Conditions

The catalytic properties of mimic enzyme are affected by some environmental factors. To obtain the maximum catalytic ability of NH2-MIL-88B(Fe)@MRGO, the experimental conditions were optimized, including temperatures were changed (30–60 °C), reaction time (5–35 min), H2O2 concentration (0–25 μM) and pH (0–12) were varied to optimize the experiment condition (Fig. S2). The optimum conditions for the detection should be pH of 4, time of 20 min, temperature of 45 °C and H2O2 concentration of 10 μM.

3.4 Biomorphic Detection of H2O2

On the basis of the peroxidase-like property of NH2-MIL-88B(Fe) and its analogues, we used a simple colorimetric method to detect H2O2 as described above. Figure 4 shows the change in the absorbance intensity at 652 nm with the concentration of H2O2. The absorbance at 652 nm is gradually enhanced with the increasing concentration of H2O2 and finally reached equilibrium steady state. UV absorption of NH2-MIL-88B(Fe)@MRGO reached a plateau at the H2O2 concentration of 150 μM outperforming of NH2-MIL-88B(Fe) (300 μM), calcined NH2-MIL-88B(Fe) (400 μM) and NH2-MIL-88B(Fe)@GO(175 μM). More importantly, the gradual increase of absorbance shown a good linearity with below the saturation concentration of H2O2. Such a good linear relationship can be used as the standard curve for quantifying H2O2 in aqueous solution (Fig. S3).

Fig. 4
figure 4

Absorbance change with increasing concentration of H2O2: NH2-MIL-88B(Fe) (a) and calcined NH2-MIL-88B(Fe) (b), NH2-MIL-88B(Fe)@GO (c), NH2-MIL-88B(Fe)@MRGO (d) (Inset: The inset photograph is the color changes corresponding to the different reaction systems at varying H2O2 concentration)

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3.5 Steady-State Kinetic Assay of NH2-MIL-88B(Fe)@MRGO

To evaluate mimic enzyme catalysis of NH2-MIL-88B(Fe)@MRGO, we first carried out the steady-state kinetic assays of GO and MRGO. It is found that the catalytic reaction rates of GO and MRGO increase at the same time as the concentration of H2O2 increases (Fig. S4). The Lineweaver–Burk diagram follows the typical Michaelis–Menten mechanism, showing a good linear relationship. Km is an indicator of the affinity of the enzyme and the substrate. The Km value is inversely proportional to the affinity between the catalyst and the substrate. The Vmax and Km values shown in Table S1 indicate that graphene oxide and MRGO, as the substrate materials for the heterogeneous mimetic enzyme prepared in this paper, both exhibit a certain catalytic affinity for H2O2 and may promote NH2-MIL-88B(Fe)@GO and NH2-MIL-88B(Fe)@MRGO mimic enzyme activity.

Further, steady-state kinetic constants were investigated to elucidate further the peroxidase activity of four mimic enzymes. These constants were determined by varying the concentrations of H2O2 while keeping the concentration of TMB at a fixed concentration in detection system. Resultantly, the Michaelis–Menten constant (Km) and maximum initial rate (Vmax) are determined using a Lineweaver–Burk plot. As displayed in Fig. 5, as the concentration of TMB and H2O2 increased, the reaction rate of mimic enzyme catalysis increased simultaneously. As depicted in the inserts, The Lineweaver–Burk plots followed a typical Michaelis–Menten mechanism [43, 44]. It is known that Km is an indicator of enzyme affinity to substrates. the Km value is inversely proportional to the affinity between catalyst and substrates. Obviously, NH2-MIL-88B(Fe), calcined NH2-MIL-88B(Fe), NH2-MIL-88B(Fe)@GO, NH2-MIL-88B(Fe)@MRGO all exhibited favorable catalytic affinity toward H2O2. As seen in Table 1, the Vmax and Km values of four mimic enzymes were listed. These as-prepared mimic enzymes well-perform the samples as reported previously in mimic enzyme catalysis. Moreover, the values of Km and Vmax for the NH2-MIL-88B(Fe)@MRGO with H2O2 were calculated to be 0.0091 mM and 2.57 × 10−7 M s−1. By comparing the apparent kinetic parameters of other mimic enzyme, the values of Km NH2-MIL-88B(Fe)@MRGO was the lowest, and the Vmax was the highest [45, 46], indicating that NH2-MIL-88B(Fe)@MRGO nanoparticles has the largest affinity for substrates.

Fig. 5
figure 5

Steady-state kinetic assay and catalytic mechanism of NH2-MIL-88B(Fe) (a) and calcined NH2-MIL-88B(Fe) (b), NH2-MIL-88B(Fe)@GO (c), NH2-MIL-88B(Fe)@MRGO (d). The velocity (v) of the reaction was measured using 0.4 mg mL−1 four kind of nanoparticles in 1 mL of HAc-NaAc buffer solution (0.2 M, pH = 4) at 45 °C, respectively. The concentration of TMB was 1 mM for four mimic enzymes and the H2O2 concentration varied. The inset image is the Linear correlation between concentration of 1/H2O2 concentration and 1/velocity

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Table 1 Comparison of Michaelis–Menten constants (Km) and maximum reaction rates (Vmax)
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3.6 Glucose Detection Using NH2-MIL-88B(Fe)@MRGO

It is well-known that glucose can be oxidized in the oxidation reaction by glucose oxidase (GOx) to gluconolactone and H2O2. NH2-MIL-88B(Fe)@MRGO and H2O2 simulate the interaction of peroxidase and TMB, which is achieved by the color reaction of TMB analysis (Fig. 6). This property has been developed for the fabrication of glucose sensors with high sensitivity and selectivity [51, 52].

Fig. 6
figure 6

Illustration of the glucose detection via the decomposition of H2O2 catalyzed by NH2-MIL-88B(Fe)@MRGO

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According to the above mechanism, we built a sensitive colorimetric sensing platform for glucose detection. As presented in Fig. 7, the absorption spectra of oxide TMB increased as the concentration of glucose increased, and the absorbance was linearly correlated to glucose concentration from 20 to 800 μM. The linear regression equation is A = 0.0005902 C (μM) + 0.03285 with a reliable correlation coefficient. As calculated from the linear calibration plots, the detection limit of the constructed colorimetric sensing platform was 3.16 μM, which is comparable or even superior to those achieved by using other colorimetric methods. Therefore, the established colorimetric sensing platform possesses prominent performance with high sensitivity and simplicity.

Fig. 7
figure 7

Linear response of the detection system to glucose using NH2-MIL-88B(Fe)@MRGO (Inset: The inset photograph is the color changes corresponding to the different reaction systems at varying glucose concentration)

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In addition, excellent peroxidase analog materials should have good peroxidase activity, but also have high selectivity to glucose. To evaluate the selectivity of the colorimetric method for glucose, control experiments were performed in the presence of glucose analog substances including 5 mM fructose, 5 mM maltose, 5 mM lactose and 5 mM sucrose (Fig. 8). The absorption intensity of glucose was much higher than its analogues; no obvious blue color is observed for other compounds except maltose. The above observations indicate that our sensing system exhibits excellent selectivity for glucose.

Fig. 8
figure 8

Selectivity analysis for glucose detection using GOx and NH2-MIL-88B(Fe)@MRGO catalyst by monitoring the relative absorbance (inset: the color change corresponding to different sample)

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A good peroxidase analogue not only has a good peroxidase activity, but also have a higher practicality for glucose. In order to evaluate the ability of NH2-MIL-88B(Fe)@MRGO to detect glucose in beverages, it was carried out in the presence of glucose mimics of 5 mM Sydney soda, 5 mM Sprite, 5 mM Coca cola, 5 mM Ice Black and 5 mM Teaminutemaid (Fig. 9). It can be seen from the figure that NH2-MIL-88B(Fe)@MRGO has a certain response to glucose in five kinds of beverages, indicating that the peroxidase analogues synthesized in this study can be used in actual samples.

Fig. 9
figure 9

Analysis for glucose detection in real samples using GOx and NH2-MIL-88B(Fe)@MRGO catalyst by monitoring the relative absorbance

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4 Conclusion

In summary, MOFs-based mimic enzymes were successfully synthesized by a simple and effective method. These mimic enzymes were functionalized with the peroxidase-mimic activity of Fe3O4, graphene, and NH2-MIL-88B(Fe), due to a huge active center for absorption and catalysis. NH2-MIL-88B(Fe)@MRGO was used for biomimetic detection of glucose. This mimic enzyme shows a high catalytic velocity (2.57 × 10−7 M s−1) and affinity (Km = 0.0091 mM) for substrates of H2O2. A limit of detection (LOD) for glucose reaches 3.16 μM. Such MOFs-based offers a unique platform for the development of highly stable and efficient mimic enzymes in catalysis, biosensing and medical diagnosis.