MIL‐53(Fe): A Metal–Organic Framework with Intrinsic Peroxidase‐Like Catalytic Activity for Colorimetric Biosensing

Showing MOFs' true colors: An iron-based metal–organic framework, MIL-53(Fe), is explored as an enzyme mimic with intrinsic peroxidase-like activity. MIL-53(Fe) can catalyze the oxidation of different peroxidase substrates in the presence of H2O2 (see graphic; TMB=3,3′,5,5′-tetramethylbenzidine, OPD=o-phenylenediamine), providing a new and simple colorimetric detection of hydrogen peroxide and ascorbic acid. Owing to both the enhanced instrumental transduction and the potential for direct visual readout, colorimetric biosensing has drawn intense attention in biological science and analytical chemistry. It offers the advantages of simplicity, rapidity, and cheapness as well as the fact that there is no requirement for any sophisticated instrumentation. As a basis for this technique, colorimetric sensors that signal analyte interaction through a change in color are undoubtedly crucial for its successful implementation. To this end, biosensors based on enzyme-mimetic inorganic materials have emerged as a new class of ideal and important colorimetric detection tools for biosensing, owing to their high stability, easy preparation, controllable structure and composition, and tunable catalytic activity.1 So far, a number of inorganic materials with peroxidase-like activity, including oxides,2 metals,3 sulfides,4 carbon,5 and polyoxometalates6 have been successfully exploited. Metal–organic frameworks (MOFs) are an intriguing class of porous crystalline inorganic–organic hybrid materials built from metal ions and polyfunctional organic ligands and have attracted increasing attention in recent years, owing to both fundamental scientific interest and attractive applications.7 Particularly, the fascinating features, which include structural diversity, flexibility and alterability, intrinsic porosity, and desired chemical functionality, endow them with great promise in a variety of fields such as gas storage, separation, drug delivery, bioimaging, and catalysis.8 Very recently, great effort has been made to provide new insights into the application of MOFs in sensing.9 Herein, we report that MIL-53 iron(III) terephthalate (MIL-53(Fe)), a typical iron-based metal–organic framework (MOF) with formula Fe(OH)(O2CC6H4CO2)⋅H2O (Scheme S1 in the Supporting Information), possesses intrinsic peroxidase-like activity, catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and 1,2,3-trihydroxybenzene (THB) in the presence of H2O2. MIL-53(Fe) as a peroxidase mimic provided a colorimetric assay for H2O2 (Scheme 1A). Moreover, an inhibition effect was induced by ascorbic acid (AA) on the oxidation of OPD catalyzed by MIL-53(Fe) in the presence of H2O2, leading to a simple colorimetric method for the detection of AA (Scheme 1B). Schematic illustration of colorimetric detection of A) H2O2 and B) AA by using MIL-53(Fe) as a peroxidase mimetic. Metal–organic framework MIL-53(Fe) was prepared by a facile one-pot solvothermal method using DMF as solvent. The structure and morphology of MIL-53(Fe) were identified by powder X-ray diffraction (PXRD), Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy dispersive X-ray (EDX) spectroscopy and UV/Vis absorption spectroscopy. The PXRD pattern of MIL-53(Fe) (Figure 1 A) shows that the as-obtained sample was crystalline, and the diffraction peaks were coincident with the previously reported MOF MIL-53 as well as the simulated one.10 The SEM image (Figure 1 B) shows that MIL-53(Fe) mainly consists of pseudo sphere-like aggregates with a size of 200–800 nm. The chemical composition determined by the EDX spectrum (Figure S1 in the Supporting Information) reveals that the C, Fe, and O elements coexist in MIL-53(Fe). The EDX elemental mapping (Figure S2 in the Supporting Information) further confirmed these elements were uniformly distributed in MIL-53(Fe). FTIR spectroscopy (Figure 1 C) shows the characteristic vibration bands of the framework OCO groups, reflecting the presence of the dicarboxylate linker in MIL-53(Fe). The diffuse reflectance UV/Vis spectrum of MIL-53(Fe) (Figure 1 D) shows a strong optical absorption band at 220–350 nm, attributed to ligand-to-metal charge transfer (LMCT).11 The weight losses (Figure S3 in the Supporting Information) of MIL-53(Fe), as determined by thermogravimetric analysis (TGA), are well within the expected range. A) PXRD patterns of a) the simulated MIL-53(Fe) created from CIF in ref. 10a, b) MIL-53(Fe) before the catalytic reaction, and c) MIL-53(Fe) after the catalytic reaction. B) SEM image of MIL-53(Fe). C) FTIR spectrum of MIL-53(Fe). D) UV/Vis diffuse reflectance spectrum of the as-prepared MIL-53(Fe). The peroxidase-like activity of MIL-53(Fe) was evaluated by the catalytic oxidation of peroxidase substrate TMB in the presence of H2O2. As shown in Figure 2 a and b, in the absence and presence of H2O2, a colorless TMB solution was observed, which displayed a negligible absorption in the range 350 to 800 nm, indicating that no oxidation reaction occurred in the absence of MIL-53(Fe). In contrast, MIL-53(Fe) was highly active in catalyzing the oxidation of TMB substrate by H2O2. The addition of MIL-53(Fe) produced a typical deep-blue color in the reaction mixture, and the solutions exhibited intense characteristic absorbance at 369 and 652 nm (Figure 2 c), bands that are ascribed to the charge-transfer complexes derived from the one-electron oxidation of TMB,12 similar to the phenomena observed for the commonly used horse radish peroxidase (HRP) enzyme.13 As Fe3+ ions are Fenton-like reagents, they could also catalyze TMB oxidation in the presence of H2O2 (Figure S4 in the Supporting Information).14 The concentration of iron ions in the supernatant of MIL-53(Fe) solution was detected by using the colorimetric method.1b, 15 In the pH range of 2.0–6.0, the absorbance at 508 nm for the supernatants of the MIL-53(Fe) solution can be ignored as it indicates that the iron ions scarcely leached from MIL-53(Fe).1b Furthermore, the phase structure of MIL-53(Fe) after the peroxidase reaction remained unchanged (Figure 1A-a and Figures S5 and S6 in the Supporting Information). All these observations indicate that MIL-53(Fe) possesses peroxidase-like activity for oxidation of TMB in the presence of H2O2. Moreover, the peroxidase-like activity of MIL-53(Fe) was confirmed by catalytic oxidation of other peroxidase substrates such as OPD16a–16c and THB9h, 16c in the presence of H2O2; these reactions could also produce the typical color changes (Figure S7 in the Supporting Information). UV/Vis spectra of a) the TMB solution, b) TMB and H2O2, c) TMB, H2O2, and MIL-53(Fe), d) MIL-53(Fe) suspension in a pH 4.0 acetate buffer at 40 °C for 30 min. [TMB]: 0.19 mM, [H2O2]: 38 mM, [MIL-53(Fe)]: 0.038 mg mL−1. Inset shows corresponding photographs. The peroxidase-like catalytic activity of MIL-53(Fe) was, therefore, further investigated by selecting the substrates TMB and H2O2 as a model reaction system. The peroxidase-like activity of MIL-53(Fe) was measured while varying the pH from 2.0 to 10.0, the temperature from 10 °C to 60 °C, the H2O2 concentration from 0.95 μM to 545 mM, and the catalyst concentration from 0.1 to 4 mg mL−1(Figures S8–11 in the Supporting Information). The catalytic activity of MIL-53(Fe) was found to be closely dependent on pH, temperature, H2O2 concentration, and catalyst concentration. The optimal conditions are approximately pH 4.0, 40 °C, and 480 μM H2O2, conditions similar to those previously reported for nanostructure-based peroxidase mimetics and HRP.14a, 17 The steady-state kinetic assays were carried out by changing the concentration of TMB and H2O2 in this catalytic system, the results of which indicate that the reaction catalyzed by MIL-53(Fe) obeys the typical Michaelis–Menten mechanism (Figure S12 in the Supporting Information).13 The kinetic parameters, such as the Michaelis–Menten constant (KM) and maximum initial velocity (Vmax), were obtained from a Lineweaver–Burk plot.13 The KM value of MIL-53(Fe) with H2O2 as the substrate was much lower than those of HRP and other nanomaterials-based peroxidase mimics (Tables S1 and S2 in the Supporting Information), suggesting that MIL-53(Fe) has a much higher affinity to H2O2 than HRP and other mimics. The catalytic mechanism of MIL-53(Fe) was further investigated by the detection of in situ-generated hydroxyl radicals (.OH) with a photoluminescence (PL) method.18 A gradual increase in PL intensity at about 425 nm was observed with increasing MIL-53(Fe) concentration (Figure S13 in the Supporting Information). Notably, there was no PL intensity in the absence of MIL-53(Fe). This solidly confirms that MIL-53(Fe) can catalytically activate H2O2 to produce .OH radicals, which could then react with TMB to produce a color change in the reaction.1b, 2c, 14b, 19 To further support this mechanism, the electrocatalytic activity of MIL-53(Fe)-modified glassy carbon electrodes (MIL-53(Fe)/GCE) towards the electrochemical reduction of H2O2 was studied by using their amperometric response. The reduction current increased sharply to a steady-state value for MIL-53(Fe)/GCE upon the addition of an aliquot of H2O2 (Figure S14 in the Supporting Information). The observable electrocatalytic activity could be attributed to the promotion of electron transfer between H2O2 (electron acceptor) and the electrode (electron donor). Based on the above, it is believed that the peroxidase-like activity of MIL-53(Fe) could originate from its catalytic activation of H2O2 through electron transfer to produce .OH radicals by a Fenton-like reaction.14, 19 Based on the aforementioned intrinsic peroxidase-like property of MIL-53(Fe), we developed a simple colorimetric method for the detection of H2O2 by using the MIL-53(Fe)-catalyzed colored reaction. Figure 3 shows the dependence of the absorbance at 652 nm on the concentration of H2O2 under optimal conditions (i.e., pH 4.0, 40 °C). The absorbance at 652 nm increases with increasing H2O2 concentration from 0.95 μM to 0.48 mM. A linear relationship (inset in Figure 3) between the absorbance and the H2O2 concentration between 0.95 and 19 μM (R2=0.990) was obtained, with a detection limit of 0.13 μM, which is lower than that provided by Fe3O4 nanoparticles.2b Furthermore, the color variation is obvious on visual observation (inset in Figure 3), offering a convenient approach to detect H2O2 by the naked eye even at low concentrations. A dose-response curve for H2O2 detection using MIL-53(Fe) under the optimum conditions described. Inset: linear calibration plot for H2O2 and corresponding photographs of the colored reaction mixtures for different concentrations of H2O2. In addition, we also observed that MIL-53(Fe) could catalyze the oxidation of the other peroxidase substrates, such as OPD, in the presence of H2O2, resulting in a yellow–orange solution (Figure S15 in the Supporting Information) and, interestingly, we find that with the addition of a trace amount of ascorbic acid (AA), the catalytic activity of MIL-53(Fe) was greatly suppressed, subsequently yielding a light yellow–orange solution (Figure S15 in the Supporting Information). Based on these observations, we therefore designed a colorimetric method for detection of AA with this MIL-53(Fe)-based sensing system. Figure 4 A shows a typical AA concentration-response curve, which indicates that the intensity of the absorption peak at 450 nm decreases with increasing AA concentration. The corresponding calibration plot is shown in Figure 4 B. The linear detection range is estimated to be 28.6 to 190.5 μM (R2=0.992). The detection limit is estimated to be 15 μM. A) UV/Vis spectra of OPD oxidation catalyzed by MIL-53(Fe) in the presence of inhibitor AA in a pH 4.0 acetate buffer at 25 °C for 15 min ([OPD]: 2.4 mM, [H2O2]: 0.24 M, [MIL-53(Fe)]: 0.038 mg mL−1). B) Linear relationship between change in absorbance at 450 nm and concentration of AA. ΔA=A0−Ai (A0 and Ai are the absorbance at 450 nm before and after addition of AA with a concentration of i, respectively). In summary, we have demonstrated that MIL-53(Fe) possesses highly efficient intrinsic peroxidase-like activity, catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) in the presence of H2O2. MIL-53(Fe) as a peroxidase mimic provided a colorimetric assay for H2O2. Furthermore, the inhibition effect of ascorbic acid (AA) on the oxidation of OPD catalyzed by MIL-53(Fe) in the presence of H2O2 was investigated. Based on the AA-induced inhibition of the peroxidase-like activity of MIL-53(Fe), a simple colorimetric method for the detection of AA was realized. We believed that the present work could open up the possibility of utilizing MOFs as enzymatic mimics in immunoassays and biotechnology. This work was supported by National Natural Science Foundation of China (Grant No. 21103141, 21207108), Sichuan Youth Science and Technology Foundation (Grant No. 2013 JQ0012), the Applied Basic Research Program of Sichuan Provincial Science and Technology Department (Grant No. 2011 JYZ019) and the Research Foundation of CWNU (Grant No. 12B018). As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. 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