Introduction

Artificial enzymes imitate the function of natural enzymes by using alternative materials [1, 2]. Nanomaterials, such as gold nanoparticle [3], metal oxide nanoparticle [4,5,6]and metal organic frameworks [7, 8], as the alternatives of natural enzymes have been found to exhibit unexpected enzyme-like activity with advantage of high stability and low cost. These discovered artificial enzymes have been demonstrated the potential capacity of being applied in bioassay and pharmaceutical processes [9, 10]. To obtain higher enzyme-like activity, the functional assembly strategy of different components will provide new paradigms to enhance properties via the synergic effects. The bimetal nanomaterials, such as Au@Pt [11], Au@Ag [12], Au/CeO2 [13], Co/2Fe-MOF [14], with improved enzyme-like activity compared to the monometallic analogues have been reported. However, with the mind of further reducing the cost and simplifying the synthesis process, the rational design and synthesis of multicomponent artificial enzymes with enhanced catalytic performance is extremely necessary.

Metal organic gels (MOGs), an analogues of MOFs, formed by self-assembly of metal ions and organic linkers have attracted considerable interest in many fields due to their attracting features, including high surface areas, tunable porosities and inherently present open metal sites [15]. Taking the biggest advantages of easy preparation under mild conditions and good compatibility, so many kinds of MOGs have been prepared and widely used in catalysis [16,17,18], sensing [19], gas storage [20], drug delivery [21] and environmental pollution abatement [22]. In our previous work, Fe-based MOGs with excellent oxidase-like activity was successfully synthesized and applied for the detection of dopamine by catalysis of luminol chemiluminescence [23]. However, as far as we know, the bimetallic organic gel with enzyme-like activity has not been reported up to now. Considering the simplified synthesis steps, low cost and good compatibility, the MOGs are a potential candidate for the preparation of multicomponent nanozymes system.

The Cu/Co bimetallic organic gel (Cux/Coy-MOG) prepared by simple mixing Co2+/Cu2+ mixture metal ions and 2,4,6-Tri(4-carboxyphenyl)-1,3,5-triazine (H3TATAB) in a mild condition was found to show enhanced peroxidase-like activity for the first time (Scheme 1). The synergetic mechanism of redox cycle that between the Co(III)/Co(II) and Cu(II)/Cu(I) pairs was based on the X-ray photoelectron spectroscopy (XPS) and cyclic voltammetry measurements. Additionally, based on this artificial enzymatic reaction system, it was successfully applied to detect H2O2 and glucose with satisfactory results. This work may provide a highly promising strategy for reasonable design and synthesis of bimetallic-based nanozymes with high performance via simple process.

Scheme 1
scheme 1

Schematic illustration of synthesis of Cux/Coy-MOG bimetallic gel and the catalytic activity towards the reaction of H2O2 with terephthalic acid

Full size image

Experimental section

Chemicals and apparatus

2,4,6-Tri(4-carboxyphenyl)-1,3,5-triazine (H3TATAB) was purchased from Na Qian Chemistry Co., Ltd. (Shanghai, China, http://xinxiang62376378.cn.zhsho.com/). Terephthalic acid (TPA) was obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China, http://aladdin.company.lookchem.cn/). Cupric (II) chloride dihydrate (CuCl2·2H2O), cobalt (II) chloride hexahydrate (CoCl2·6H2O) were obtained from Tianjin Hygain Chemical (Group) Co., Ltd. (Tianjin, China, http://tianjinhg2008.51pla.com/). Glucose, L-galactose, pectinose, mannose, fructose, maltose, and sucrose, and 30% (v/v) H2O2 were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China, https://www.instrument.com.cn/netshow/SH101458/). All reagents were analytical grade and commercial, and without further purification. The serum samples were kindly provided by Southwest university hospital.

An S-4800 scanning electron microscope (SEM) (Hitachi, Japan) was used to imaging the morphology of Cux/Coy-MOG. Transmission electron microscopic (TEM) characterization was performed on JEOL JEM-1200EX TEM instrument (JEOL, Japan). The xerogels were obtained by a Cool safe 110–4 freeze-drying apparatus (Labogene, Denmark). Powder X-ray diffraction (PXRD) patterns were collected on a D8ADVANCE X-ray diffractometer (Bruker, Germany) with Cu Kα radiation (λ = 1.5406 Å) at a scan rate of 3.00o min−1. Cyclic voltammetric measurement was performed on a CHI 660E electrochemical workstation (CH Instruments, Shanghai, China). A conventional three-electrode cell was constructed with modified glassy carbon electrode (GCE) as working electrode, Pt wire as counter electrode and Ag/AgCl electrode as reference electrode. F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan) was used for fluorescence measurement. The instrument settings as follows: λex = 315 nm (slit 5 nm), λem = 446 nm (slit 5 nm), PMT detector voltage = 400 V. A pH-510 digital pH-meter with a combined glass electrode (California, USA) was performed to test pH. The water used in all the experiments was purified by a Milli-Q system (Millipore, USA).

Preparation of Cux/Coy metal-organic gels (MOG)

Typically, 200 mg (0.46 mmol) of H3TATAB was dissolved in 10 mL ultrapure water (containing 200 μL triethylamine (TEA)) to prepare stock solution. The stock solution of Co2+ and Cu2+ were prepared by dissolving CuCl2·2H2O (0.46 mmol, 79 mg) and CoCl2·6H2O (0.46 mmol, 109 mg) in 10 mL ultrapure water, respectively. For the synthesis of MOGs, mixture metal ion solution with variable ratio of Cu/Co (from 1:0 to 0:1, while maintaining the total concentration was 0.046 M) was simply mixed with equal volume H3TATAB (0.046 M). After standing 15 min at room temperature, a series of Cux/Coy-MOG were obtained. To quantify it expediently, the Cux/Coy-MOG were further treated by lyophilization to obtain xerogel.

Preparation of serum samples

The samples were first treated by spin dialysis at 10000 rpm for 45 min, and then the eluents were diluted with phosphate buffer (PB, 10 mM, pH 7.0) before measurements.

Determination of hydrogen peroxide and glucose

The Cu0.5/Co0.5-MOG (0.2 mg·mL−1, 50 μL), TPA (20 mM, 50 μL) and H2O2 with different concentrations were orderly added into 300 μL of phosphate buffer (pH 7.0), and the mixture was diluted with ultrapure water to a volume of 500 μL. After incubating at 45 °C for 20 min, fluorescence spectra were measured under the excitation wavelength of 315 nm.

The detection of glucose was carried out as follows: Typically, GOx (1 mg·mL−1, 50 μL) and 50 μL glucose with different concentrations were added into 300 μL of phosphate buffer (pH 7.0), and incubated at 37 °C for 20 min. TPA (20 mM, 50 μL) and Cu0.5/Co0.5-MOG solution (0.2 mg·mL−1, 50 μL) were added to the above glucose reaction solution for another 2 h at 45 °C. The resulting reaction solution was measured by using F-2500 fluorescence spectrometer under the excitation wavelength of 315 nm and detecting the emission intensity at 446 nm. For the detection of glucose in serum, the serum eluents were used instead of glucose solution.

Results and discussion

Choice of materials

As previously reports, the peroxidase-like activities of many nanomaterials, especially noble metal nanoparticles, may be negatively affected by non-specific absorption [2]. Besides, noble metal nanomaterials are prone to aggregate in complex systems, thus reducing their catalytic activity [6, 24]. What’s more, the preparation process is usually costly and complicated. Therefore, it is necessary to find a high-efficiency artificial nanoenzyme with low-cost and simple preparation process. MOG is a new kind of metal-organic hybrid materials, which can be prepared by simply mixing the metal ions with the ligand. Furthermore, the catalytic activity can be tuned by changing the metal ions. In our research, the common transition metal ions (such as Cr3+, Mn2+, Fe3+, co2+, Ni2+, cu2+, Zn2+) had been applied for the preparation of MOG. However, only cu2+, co2+ and Fe3+ can form stable gel with H3TATAB. The comparation of peroxidase-like activity of the MOGs indicated the Cu(II)/Co(II) bimetallic organic gel shows the best catalytic performance (Fig.S1). Therefore, Cu(II)/Co(II) bimetallic organic gel was selected as a peroxidase-mimetic to further explore their high catalytic activity and application.

Characterization of material

A series of Cux/Coy-MOG can be formed in a wide range molar ratio of Cu2+/Co2+, demonstrating Cux/Coy-MOG displays good compatibility (Fig. S2a-k). Taking Cu0.5/Co0.5-MOG as an example, the morphology of the MOGs was firstly examined by SEM and TEM (Fig. 1 a-f). It clearly revealed the MOGs were constructed by nanofibers. The uniform distribution of the elements C, O, N, Co and Cu in the Cu0.5/Co0.5-MOG nanofibers were evidenced by the elemental mapping (Fig.1g). There were no obvious diffraction spots except diffraction ring in selected area electron diffraction (SAED) pattern, indicating the amorphous structure of Cu0.5/Co0.5-MOG (Fig. 1h). The Brunauer-Emmett-Teller (BET) isotherm disclosed that the as-fabricated MOGs exhibit typical type-III isotherm characteristics of microporous materials (Fig. S3a, b). Furthermore, it also shows that the addition of cobalt ions reduced the BET surface area to a certain extent but with similar aperture distribution. The thermogravimetric analysis (TGA) revealed that the Cu-MOG and Cu0.5/Co0.5-MOG show high thermal stability with slow weight losses at a broad range of temperature compared with Co-MOG (Fig. S4) [25]. From the IR spectra, the stretching vibration of C=O in H3TATAB was shift from 1697 cm−1 to 1604 cm−1, indicating that the metal ions and carboxyl groups were ligated via coordinate bond (Fig. S5) [25]. The powder XRD patterns of Cu0.5/Co0.5-MOG are like those of the monometallic Cu-MOG and Co-MOG, revealing that these materials possess similar structure(Fig. S6). Furthermore, two diffraction peaks at 22.01° and 23.84° can be observed, which are matched well with the previous reported MOGs, assigning to the π-π stacking [26, 27].

Fig. 1
figure 1

SEM images of Cu-MOG, Cu0.5/Co0.5-MOG and Co-MOG (a-c), and the corresponding TEM images (d-f); (g) the TEM and elemental mapping images of Cu0.5/Co0.5-MOG for Co, Cu, C, N and O with color superposition; (h) the SAED pattern of Cu0.5/Co0.5-MOG

Full size image

Peroxidase-like activity of the Cux/Coy-MOG

A typical TPA-H2O2 reaction was employed as a model reaction to explore the peroxidase-like activity of the Cux/Coy-MOG. As shown in Fig. 2, almost no fluorescence signal can be measured from the H2O2 + TPA system (curve a), suggesting that the reaction of H2O2 and TPA can hardly happen in the absence of enzyme. The weak fluorescent was observed in the presence of Co-MOG (curve b), showing that Co-MOG had a weak mimic enzyme activity. The high catalytic activity can be observed in Cu-MOG and Cu0.5/Co0.5-MOG enzymatic reaction system, demonstrating the Cu(II) is the major catalytic activity center. Moreover, the peroxidase like activity of the Cu/Co-MOGs with various molar ratios of Cu/Co was also evaluated (Fig. S7a, b). It clearly shows that the catalytic activity highly depended upon the Cu/Co ratio. Particularly, the Cu0.5/Co0.5 is the optimum molar ratio to achieve the best peroxidase-like activity.

Fig. 2
figure 2

Fluorescence spectra of (a) H2O2 + TPA, (b) H2O2 + TPA + Co-MOG, (c) H2O2 + TPA + Cu-MOG and (d) H2O2 + TPA+ Cu0.5/Co0.5-MOG. The concentrations of H2O2, TPA and the MOGs were 8μM, 2mM, 20 mg·L−1, respectively; insert showed the amplified of (a) and (b). λex: 315 nm; λex:446 nm; slits:5nm; voltage:400V

Full size image

As we know, non-fluorescent TPA can be oxidized by •OH into the 2-hydroxyterephthalic acid (TAOH) which shows a strong fluorescence with the maximum excitation/emission at 315/446 nm. Therefore, the thiourea as •OH scavenger and electron spin resonance (ESR) study were applied as a direct method to verify the production of •OH (Fig. S8a, b). The results showed that the the fluorescence signal of TAOH can be decreased more than 95% by the presence of 2mM of thiourea, and the the typical quartet characteristic peak in ESR spectrum with relative intensities 1:2:2:1 was observed, indicating that Cu0.5/Co0.5-MOG can induce •OH production by catalysing H2O2.

Mechanism exploration

Based on the above experimental results, the bimetallic Cu0.5/Co0.5-MOG shows higher peroxidase-like activity than Cu-MOG and Co-MOG. To explore the mechanism that able to justify the superior performance of Cu0.5/Co0.5-MOG, the X-ray photoelectron spectroscopy (XPS) was performed to characterize the change of valence state of metal component before and after reacting with H2O2 (Fig. 3a). The high-resolution Cu2p XPS spectrums of Cu0.5/Co0.5-MOG before and after treating with H2O2 (Fig. 3b, c) both show main peaks at roughly 934.5 eV and 954.3 eV, which originated from the Cu(II) in Cu2p-O bonds. [24] Besides, the Cu LMM Auger spectra were performed to distinguish the valence states of the Cu species (Fig. 3d). It shows an obvious shift of Auger peak from 570.6 eV to 571.8 eV after H2O2 treatment, confirming that the formation of Cu(I) in the Cu0.5/Co0.5-MOG after reacting with H2O2. [28, 29] The XPS patterns and data analysis of Co species were displayed in Fig. 3e, f and Table S1, respectively. The spin-orbit splitting ΔE(2p1/2-2p3/2) was changed from 15.9 eV to 15.4 eV, and the significantly reduced Σsat/ICo2p3/2 value were observed after treating with H2O2, demonstrating the presence of Co(III) component [30, 31].

Fig. 3
figure 3

a High resolution XPS spectra of Cu0.5/Co0.5-MOG before (black line) and after (red line) treat with H2O2. Wide spectra and high-resolution spectra of (b) Cu2p of Cu0.5/Co0.5-MOG without H2O2 treatment, (c) Cu2p of Cu0.5/Co0.5-MOG after H2O2 treatment, (d) Cu LMM Auger spectra of Cu0.5/Co0.5-MOG before (red line) and after (black line) treat with H2O2, (e) Co2p of Cu0.5/Co0.5-MOG without H2O2 treatment, (e) Co2p of Cu0.5/Co0.5-MOG after treat with H2O2

Full size image

According to previous reports, the catalytic mechanism of Cu(II) and Co(II) can be described in the following reactions: [32,33,34]:

$$ \mathrm{Co}\left(\mathrm{II}\right)+{\mathrm{H}}_2{\mathrm{O}}_2\to \mathrm{Co}\left(\mathrm{II}\mathrm{I}\right)+\bullet \mathrm{OH}+{\mathrm{O}\mathrm{H}}^{-} $$
(1)
$$ \mathrm{Cu}\left(\mathrm{I}\mathrm{I}\right)+{\mathrm{H}}_2{\mathrm{O}}_2\to \mathrm{Cu}\left(\mathrm{I}\right)+\bullet {\mathrm{O}}_2\mathrm{H}+{\mathrm{H}}^{+} $$
(2)
$$ \mathrm{Cu}\left(\mathrm{I}\right)+{\mathrm{H}}_2{\mathrm{O}}_2\to \mathrm{Cu}\left(\mathrm{I}\mathrm{I}\right)+\bullet \mathrm{OH}+{\mathrm{O}\mathrm{H}}^{-} $$
(3)

As described above, a synergetic catalytic mechanism of Cu0.5/Co0.5-MOG was proposed as follows: 1) once H2O2 was added, •OH radicals were generated by active sites including Cu(II) and Co(II) on the catalyst surface, resulting the generation of Cu(I) and Co(III); 2) according to cyclic voltammogram and eq. (6) (Fig. S9 and Table 1), the Cu(I) oxidation by Co(III) is thermodynamically feasible, which is conducive to the redox cycles of Cu(II)/Cu(I) and Co(III)/Co(II) via single electron transfer. This process can accelerate the interfacial electron transfer within Cu0.5/Co0.5-MOG. Therefore, the synergetic effect caused by the endogenous redox cycle of Cu(II)/Cu(I) and Co(III)/Co(II) pairs is benefit for the more •OH generation (Fig. 4).

Table 1 The reaction potentials from cyclic voltammogram curve of Cu0.5/ Co0.5-MOG
Full size table
Fig. 4
figure 4

Possible mechanism for Cu0.5/Co0.5-MOG as artificial enzyme

Full size image

Hydrogen peroxide and glucose detection

As we all know, the enzymatic activity greatly depends on the reaction condition, such as concentration of catalyst and substrate, pH and temperature. The optimization of experimental conditions is displayed in Fig. S10. Under the optimal reaction conditions, the typical fluorescence response was observed in the presence of different concentrations H2O2, and the fluorescence intensity versus hydrogen peroxide concentration was linear over the range from 1.0 μM to 10 μM. The linear regression equation was F = 100.54 c-81.72 (R2 = 0.997) (where F is the fluorescence intensity and c is the concentration of H2O2, respectively.) The limit of detection was approximately 81 nM (S/N = 3) (Fig. 5a, b). As shown in Fig. S11, it clearly demonstrated that the common ions did not affect the detection of H2O2. Moreover, the Cu0.5/Co0.5-MOG was used for determination of glucose combining with glucose oxidase. As Fig. 5c shown, the fluorescence intensity increased markedly along with the increase of glucose concentration. Good linear relationship was obtained in the range of 0.5 μM to 120 μM. The linear regression equation was F = 5.453 c-0.2424 (R2 = 0.994) and the limit of detection was calculated to be 0.33 μM (S/N = 3) (Fig. 5d). The selectivity experiment was also carried out with a satisfactory result (Fig. S12). The analytical performance of the method was also compared with other methods based on peroxidase-mimics reported in the literature (Table S2). The present method showed a good linear range and high sensitivity, and it realized the detection of H2O2 and glucose at the sub-μM levels.

Fig. 5
figure 5

a Fluorescence spectra of Cu0.5/Co0.5-MOG + TPA with various concentrations of H2O2 from 0 to 400 μM in phosphate buffer (pH 7.0); (b) Plot of the fluorescence intensity of Cu0.5/Co0.5-MOG + TPA against the concentrations of H2O2, insert: the linear calibration plot for H2O2; (c) Fluorescence spectra of Cu0.5/Co0.5-MOG + TPA + GOx with various concentrations of glucose from 0 to 120 μM; (d) Linear calibration plot for glucose. λex: 315 nm; λex:446 nm; slits:5nm; voltage:400V

Full size image

To confirm the precision and reliability of this method, the standard addition method was performed. The spiked samples were obtained by adding a certain amount of glucose to the dilute serum. Table S3 shows the acceptable recovery rates (98%–108%). This demonstrates that the method can be used for the detection of glucose in serum samples. Besides, the reproducibility of Cu(II)/Co(II) bimetallic organic gel in the glucose oxidase/glucose system was investigated (Fig. S13). The results clearly indicated that no significant loss of activity in the seven successive using, suggesting that the Cu(II)/Co(II) bimetallic organic gel possesses long-term stability and reproducibility. On the other hand, the morphology of Cu(II)/Co(II) bimetallic organic gel in different systems has been studied by SEM (Fig. S14). The results show the morphology of Cu(II)/Co(II) bimetallic organic gel has no serious changes in different systems.

Conclusions

In summary, the Cu0.5/Co0.5-MOG with enhanced peroxidase-like activity has been designed and synthesized successfully. The excellent performance was attributed to the synergistic effect of endogenous redox cycle that between Cu(II)/Cu(I) and Co(III)/Co(II) pairs, leading more generation of •OH. Subsequently, the Cu0.5/Co0.5-MOG was successfully applied to fluorescent detection of glucose in serum. We believe that this study would light a new beacon for the design and synthesis of peroxidase-like enzyme, and may stimulate new development in this field. The potential applications of Cux/Coy-MOG in bioassay, environmental remediation, and catalysis are also expected to be broadened.