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Protein-Metal Organic Framework Hybrid Composites with Intrinsic Peroxidase-like Activity as a Colorimetric Biosensing Platform Yuqing Yin, Chen Ling Gao, Qi Xiao, Guo Lin, Zian Lin, Zongwei Cai, and Huang-Hao Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09893 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 5, 2016

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ACS Applied Materials & Interfaces

Protein-Metal Organic Framework Hybrid Composites with Intrinsic Peroxidase-like Activity as a Colorimetric Biosensing Platform Yuqing Yin,† Chenling Gao,† Qi Xiao,† Guo Lin,† Zian Lin,†* Zongwei Cai‡ and Huanghao Yang†

† Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China ‡ Partner State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong, SAR, P. R. China

Corresponding author: Zi-An Lin; Postal address: College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, China Fax: +86-591-22866165 E-mail: [email protected] (Z.A. Lin);

KEYWORDS: Hemeproteins, Metal-organic framework, Peroxidase mimetics, Colorimetric detection, Hydrogen peroxide, Phenol. 1 ACS Paragon Plus Environment


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ABSTRACT Artificial enzyme mimetics have received considerable attention because natural enzymes have some significant drawbacks, including enzyme autolysis, low catalytic activity, poor recovery and low stability to environmental changes. Herein, we demonstrated a facile approach for one-pot synthesis of hemeprotein-metal organic framework hybrid composites (H-MOFs) by using bovine hemoglobin (BHb) and zeolitic imidazolate framework-8 (ZIF-8) as a model reaction system. Surprisingly, the new hybrid composites exhibits 423% increase in peroxidase-like catalytic activity compared to free BHb. Taking advantages of the unique pore structure of H-MOFs with high catalytic property, a H-MOFs-based colorimetric biosensing platform was newly constructed and applied for the fast and sensitive detection of hydrogen peroxide (H2O2) and phenol. The corresponding detection limits as low as 1.0 µM for each analyte with wide linear ranges (0-800 µM for H2O2 and 0-200 µM for phenol) were obtained by naked-eye visualization. Significantly, sensitive and selective method for visual assay of trace H2O2 in cell and phenol in sewage was achieved with this platform. The stability of H-MOFs was also examined and excellent reproducibility and recyclability without losing in its activity were observed. In addition, the general applicability of H-MOFs was also investigated by using other hemeproteins (horseradish peroxidase, and myoglobin) and the corresponding catalytic activities were 291% and 273% enhancement, respectively. This present work not only expands the application of MOFs, but also provides an alternative technique for biological and environmental sample assay. 2 ACS Paragon Plus Environment

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INTRODUCTION Hemeproteins are an immensely diverse classes of metallo-enzymes in nature and are prevalent in all organisms.1-4 This kind of proteins, including peroxidases, cytochromes, hemoglobins, and myoglobins, has diverse biological functions such as oxygen transportation,5 electron transfer,6 and catalysis.7-9 As the active center of hemeproteins, heme (iron porphyrin derivative) shows the peroxidase-like activity that is similar to the peroxidase enzyme.8-12 However, direct application of these hemeproteins as oxidation catalyst in aqueous solution remains a great challenge due to the low stability and recovery, sensitivity of catalytic activity in environmental changes, as well as time-consuming preparation and purification. Therefore, development of heme enzyme mimetics with peroxidase-like activity is highly desirable. Generally, there are two strategies for construction of heme enzyme mimetics. One is to protect the heme center against damage by modification of the porphyrin to produce dendrimers

13,14

or molecular crystals.15,16 The other is to load the heme on

the various supports, such as carbon materials,17-19 hydrogels,20,21 zeolites22 and metal/metal oxide nanoparticles,23-25 which show catalytic activity similar to that found in natural peroxidases, and have been successfully applied as peroxidase mimic for the analysis of small molecules. Metal-organic frameworks (MOFs), as an interesting class of hybrid nanomaterials built from metal ions and organic ligands, have attracted immense 3 ACS Paragon Plus Environment


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attention in recent years.26-28 These highly ordered crystalline materials with superior properties, such as ultrahigh surface areas, tuneable pore sizes, and easy surface modification. These attractive features make MOFs highly promising in the field of gas storage, chromatographic separation, and industrial catalysis.28-32 In the past few years, many achievements on catalytically active guest species (e.g. organometallic compounds and metalloporphyrins) encapsulated into porous MOFs have been made. For example, a variety of MOFs including MIL-53 iron ( Ⅲ ) terephthalate (MIL-53(Fe)),33 MIL-68 and MIL-100,34 Zr-MOFs,35 and Cu-MOFs36 were developed based on the intrinsic activity of inorganic materials. However, these MOFs showed relatively low catalytic activity, sensitivity and selectivity, which hinder its wide applications in bioanalysis. Recently, significant efforts have been made to develop protein encapsulated MOFs with the demand of biological applications. Lykourinou et al.37 has demonstrated for the first time that microperoxidase-11 could be successfully immobilized into mesoporous MOFs containing nanoscopic cages, and the resultant protein-encapsulated MOFs showed good enzymatic catalysis performances. Li et al.38 recently developed organophosphorus acid anhydrolase (a nerve agent detoxifying enzyme)-encapsulated Zr-MOFs based upon the similar synthetic principle, which displayed good catalytic performance, in addition to high protein loading capacity, good thermal and loner-term stabilities. Although some successes have been achieved,38 synthesis of protein-encapsulated MOFs remains a continuous challenge due to the large size of protein molecules compared to the much smaller 4 ACS Paragon Plus Environment

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pore of most of the common MOFs. Additional simple synthetic approaches are highly desirable. Until recently, a significant breakthrough in one-pot synthesis of protein-embedded MOFs was achieved by Lyu et al.39 In this work, protein was directly embedded in MOFs by a facile coprecipitation method and the as-prepared protein-embedded MOFs exhibited high biological activity compared with free protein in solution. Nevertheless, the advantages of protein-MOFs hybrid composites and the attractive synthetic approach have not yet been fully demonstrated. Further development is very necessary to address the merits and explore new applications of this type of hybrid composite materials. The employment of protein-inorganic hybrid composites with high catalytic activity for visual colorimetric bioanalysis has gained increasing interest due to the simplicity and the low cost of this type of assay.40-42 For instance, Lyu et al.39 recently developed a facile visual colorimetric method for the sensitive determination of trace explosive organic peroxides in solution by using cytochrome c (Cyt c)-embedded MOFs hybrid composites as enzyme mimetics, the successful application demonstrated the feasibility of the protein-MOFs as a colorimetric platform. Inspired by this result, herein, we developed a facile approach for the preparation of protein-embedded MOFs by using bovine hemoglobin (BHb) as the organic component and zeolitic imidazolate framework-8 (ZIF-8) as the inorganic component, respectively. The as-prepared ZIF-8@BHb hybrid composites via self-assembly exhibited good stability and high catalytic activity. Based upon it, a novel ZIF-8@BHb hybrid composites based colorimetric platform was builted and applied 5 ACS Paragon Plus Environment


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for visual detection of hydrogen peroxide (H2O2) and phenol. The results demonstrated that the hybrid composites exhibited both rapid response and sensitivity for the detection of analytes than the free BHb.

EXPERIMENTAL SECTION Reagents and Materials. Zinc nitrate hexahydrate (Zn(NO3)2·6H2O), phenol, and amino acids were purchased from Sinopharm Chemical Reagent, Co., Ltd (Shanghai, China). 2-Methylimidazole (HMeIM) was obtained from J&K Chemical Ltd (Shanghai, China). Horseradish peroxidase (HRP), myoglobin (Mb) and BHb (Mw=68000, pI=6.7) were the product of Shanghai Lanji Co. Ltd. (Shanghai, China). 3,3′,5,5′-Tetramethylbenzidine (TMB) was get from Yuanye Biotechnology Co. Ltd (Shanghai, China). H2O2 (30%, w/v) was purchased from Shantou Xilong Chemical Factory (Guangdong, China). 4-Aminoantipyrine (AAP) was purchased from Shanghai Dibai Chemicals Technology Co., Ltd (Shanghai, China). Other chemicals were of analytical grade or better. Industrial wastewater was collected from Lianjiang Sewage Treatment Plant, which was stored at 4℃ before use. Cultured liver cell was donated from Fujian Health College and stored at -20℃ before analysis. Deionized water was prepared using a Millipore Milli-Q Plus system (Millipore, Milford, MA).

Preparation of ZIF-8@BHb Hybrid Composites. 25 mL of Zn(NO3)2 (10 mM) was mixed with 25 mL of HmeIM (400 mM), and degassed in an ultrasonic bath for 10 min. Subsequently, 400 µL of BHb (50 mg/mL) was added to the above solution and sonicated for another 10 min. After that, the mixture was further incubated at room temperature for 10 h without stirring. The product (denoted as 6 ACS Paragon Plus Environment

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ZIF-8@BHb hybrid composites) was obtained by centrifugation, washed with deionized water, and then dried under vacuum at room temperature. Similarly, the ZIF-8@HRP and ZIF-8@Mb hybrid composites were also prepared by replacing BHb with HRP (50 mg/mL) and Mb (50 mg/mL), respectively.

BHb Activity Test. The catalytic activity of BHb embedded in ZIF-8 was measured by using TMB as the chromogenic substrate in the presence of H2O2.33,35 In brief, 32.7 µg of ZIF-8@BHb hybrid composites was added to 440 µL Na3Cit buffer solution (0.2 M, pH 4.0) containing 1.0 mM TMB and 3 mM H2O2 for different reaction time. After centrifugation, the supernatant was detected by UV-Vis spectrophotometer at the wavelength of 650 nm. The activity units were calculated according to the previous description.41,43 and the catalytic activity of BHb was calculated by the following equation:

BHb Activity (TMB / mg ) =

ε 650nm

∆A 650nm / min × C BHb(mg / mL ) × V BHb(mL)

(1)

Where △A650nm is the change of TMB absorbance values, ε650nm is molar extinction coefficients of TMB (ε650nm=0.358 µM-1·cm-1), CBHb represents the concentration of BHb, and VBHb is the volume of BHb. Similarly, the activity of free BHb in solution was also detected based upon the above mentioned procedures except for using an equivalent amount of BHb (3.5 µg) as a substitute for the ZIF-8@BHb hybrid composites. To further evaluate the affinity and reaction rate of the ZIF-8@BHb hybrid composites for the substrate, Michaelis-Menten kinetics was studied. In brief, 32.7 µg

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of ZIF-8@BHb composites was added to the solution containing 440 µL of Na3Cit buffer solution (0.2 M, pH 4.0), 3 mM H2O2 and TMB with different concentrations (0.2-1.0 mM). After reaction for 5 min, the supernatant was detected by UV-Vis spectrophotometer. The reaction process followed the enzymatic dynamic regulation of the Michaelis-Menten equation, and was detected by UV-Vis spectrophotometer in kinetic mode: 44

1

ν0

=

κm 1 × (C + ) νm κm

(2)

Where V0 and Vm represent the initial velocity and the maximal reaction velocity, respectively. C and Km are the concentration of substrate and the Michaelis constant accordingly.

H2O2 and Phenol Detection Test. The detection of H2O2 with ZIF-8@BHb hybrid composites was carried out as follows: different concentration of H2O2 was added to 1.0 mL phosphate buffered solution (PBS, 0.1 M, pH 7.4) that contained 200 µg ZIF-8@BHb hybrid composites, 4.0 mM AAP and 1.0 mM phenol, and subsequently incubated for 5 min at room temperature. Then, the absorbance of the supernatant was detected at the wavelength of 505 nm after centrifugation. Similarly, the detection of phenol was performed according to the above description in the presence of 1.0 mM H2O2.

Real Sample Preparation and Analysis. For determination of H2O2 in cells, the cultured liver cell was extracted according to the previous report by Borthwick et al.45 Then, 8000 cells was added to 100 µL PBS (0.1 M, pH 7.4), and destroyed by


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ultrasonic cell disrupter. After that, it was centrifuged and then the supernatant was collected for following experiments. The procedure of real sample analysis was carried out as follows: To each 1.0 mL cell sap (or wastewater), 200 µg ZIF-8@BHb hybrid composites, 4.0 mM AAP and 1.0 mM phenol (or H2O2) were added. The spiked samples were also prepared by adding different concentration of H2O2 (or phenol) in cell sap (or wastewater). After incubation at room temperature for 5 min, the sample was centrifuged, and then the absorbance of the supernatant was detected as mentioned above.

RESULTS AND DISCUSSION Preparation

and

Characterization

of

ZIF-8@BHb

Hybrid

Composites. The schematic diagram for one-pot synthesis of ZIF-8@BHb hybrid composites was depicted in Figure 1A. Specifically, ZIF-8 was firstly prepared by mixing Zn(NO3)2 solution with HMeIM, and stirring at room temperature for 10 min. Subsequently, BHb with various concentrations was added dropwise to the milky ZIF-8 solution (Figure 1B). After incubation at room temperature for 10 h, brown precipitates (ZIF-8@BHb hybrid composites) were obtained (Figure 1C). Compared with the synthetic procedure (~24 h) described by Lyu,39 the synthesis in aqueous system adopted in current work needed shorter reaction time. The assembling processes of protein-MOF hybrid composites which could be mainly influenced by protein concentration and incubation time were investigated in detail. The concentration of BHb varied from 0 to 125 mg/mL was studied and the 9 ACS Paragon Plus Environment


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weight percentages of BHb in the ZIF-8@BHb hybrid composites were measured by gravimetric analysis. The results (Table S1 in the Supporting Information) showed that the weight percentage of BHb embedded in the ZIF-8@BHb hybrid composites gradually increased from 0 to 15.98 % as the concentration of BHb increased from 0 to 125 mg/mL. Nevertheless, the encapsulation efficiency40,41 gradually increased from 0 to 59.50 % when the BHb concentration increased from 0 to 50 mg/mL, but dropped down to 37.50 % with continuous increase of BHb from 50 to 125 mg/mL. The result can be explained by the fact that BHb concentration over 50 mg/mL was enough to completely exhaust the substrate (ZIF-8).

In view of its high

encapsulation efficiency, 50 mg/mL BHb was selected as the optimized concentration for further evaluation and applications. On the other hand, reaction time is another crucial factor in formation of ZIF-8@BHb hybrid composites, and therefore the incubation time in the range of 5-36 h was also studied while keeping BHb constant. The results (data not shown) showed that the encapsulation efficiency increased slightly when the incubation time was over 10 h, indicating the complete reaction in this model reaction system. Taking the ZIF-8@BHb hybrid composites with 50 mg/mL BHb as an example, self-assembly process of ZIF-8@BHb hybrid composites was verified by means of SEM and TEM. As shown in Figure 2A(1-2), the pure ZIF-8 has a regular smooth hexagonal prism structure and no flakes were observed on the edge of the pure ZIF-8 (Figure 2A(3-4)). Although the ZIF-8@BHb hybrid composites displayed the same morphology as pure ZIF-8, its surface became rough, demonstrating the formation of the ZIF-8 crystals with amorphous structure (Figure 2B(1-2)). Moreover, TEM images 10 ACS Paragon Plus Environment

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(Figure 2B(3-4)) showed that many transparent flakes were emerged on its edge, which was consistent with the SEM results and confirmed the presence of BHb in the composites. The ZIF-8@BHb hybrid composites were characterized by Fourier transform infrared (FT-IR) spectroscopy. As presented in Figure 3A, the strong adsorption bands of ZIF-8 at 3137 and 2929 cm-1 were ascribed to the aromatic and the aliphatic C-H stretch modes, respectively. The adsorption at 1587 cm-1 could be attributed to the C=N stretch mode, and the bands in the region of 900-1330 cm-1 were thanked to plane bending of the imidazole ring. The band at 422 cm-1 was assigned as the characteristic Zn-N stretch mode. Compared to the spectra of ZIF-8, similar adsorption bands were observed in the spectrum of the ZIF-8@BHb hybrid composites, which were well matched with ZIF-8 structure.46 However, the typical band of BHb at 1600 cm-1 for −CONH was observed in the spectra of BHb and ZIF-8@BHb hybrid composites at the same time, indicating the existence of BHb in the hybrid composites. Furthermore, there were no new adsorption peaks and significant peak shift appeared in the spectra of the ZIF-8@BHb hybrid composites compared with those of BHb and ZIF-8, demonstrating that BHb was embedded via coprecipitation process, instead of covalent bonding. TGA was performed to quantitatively determine the composition of the composites. As shown in Figure 3B, a distinct weight-loss step of 22.90% up to ca. 200℃ was observed, which was attributed to the loss of guest molecules (e.g. H2O) from the cavities or unreacted species (HMeIM) from the ZIF-8@BHb hybrid 11 ACS Paragon Plus Environment


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composites. However, the ZIF-8@BHb hybrid composites displayed a gradual weight-loss profile in the range of 200−550℃, and there are approximate 11.71% weight loss in the studied range, which was consistent with the above result by gravimetric analysis. Obviously, the process can be ascribed to the weight loss of BHb in the composite. It should be noted that a long plateau was presented in the temperature range of 200–550℃, implying the good thermal stability of ZIF-8. The weight loss in both ZIF-8 and ZIF-8@BHb hybrid composites over 550℃ were associated with the thermal decomposition of ZIF-8 crystals. The crystalline structure and thermal stability of ZIF-8 with and without embedding BHb were further examined by powder X-ray diffraction (XRD), which was shown in Figure 3C. The positions and corresponding intensities of all diffraction peaks matched well by comparing the pure ZIF-8 with ZIF-8@BHb hybrid composites, and showed no changes even after heating to 400℃, validating that the ZIF-8@BHb hybrid composites were highly crystallized even after embedding BHb. Furthermore, the SEM images (Figure 3D) and TEM images (Figure S1, Supporting Information) of ZIF-8@BHb hybrid composites after calcination showed that most of transparent flakes on the edge of the ZIF-8 disappeared, but no changes occurred on ZIF-8, indicating that BHb was mainly deposited on the surface of the ZIF-8. In addition, the surface area (Figure S2, Supporting Information) and pore volume of the ZIF-8@BHb hybrid composites was determined to 811.7 m2/g and 0.43 cm3/g, which was lower than those of the pure ZIF-8 (1008.9 m2/g and 0.54 cm3/g). Apparently, embedding BHb into ZIF-8 can respond to the decrease of the surface area and pore 12 ACS Paragon Plus Environment

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volume.

Catalytic Activity and Kinetics of ZIF-8@BHb Hybrid Composites. To evaluate the feasibility of the ZIF-8@BHb hybrid composites acted as peroxidase mimic, a simple and sensitive enzyme/TMB/H2O2 reaction system was adopted.

Herein,

several

different

reaction

systems,

including

ZIF-8@BHb/TMB/H2O2, BHb/TMB/H2O2, ZIF-8/TMB/H2O2, and TMB/H2O2, were investigated individually and compared. As shown in Figure 4A, it was observed that the ZIF-8/TMB/H2O2 reaction system showed rather weak UV-Vis absorption at 650 nm, compared to any of the single/dual component in buffer (ZIF-8, TMB, H2O2, and TMB/H2O2 reaction system), indicating that the pure ZIF-8 had no catalytic activity. In contrast, the ZIF-8@BHb/TMB/H2O2 reaction system produced much stronger absorption than BHb/TMB/H2O2, and ZIF-8/TMB/H2O2 system at 650 nm. These observations firmly confirmed that the ZIF-8@BHb hybrid composites had much higher catalytic activity than free BHb and ZIF-8 for oxidation of TMB in the presence of H2O2. The catalytic activity and kinetic parameters of the ZIF-8@BHb hybrid composites were further investigated. On the basis of the different oxidation rates vs variable substrate concentrations, the Lineweaver-Burk plot was achieved with good linear relationship (Figure S3, Supporting Information), where two important kinetic parameters, including Km and Vm, could be obtained. For the TMB oxidation reaction, the Km of the ZIF-8@BHb hybrid composites was determined to 0.28 mM, which was approximately 5-fold lower than the Km of free BHb (1.02 mM), indicating that the 13 ACS Paragon Plus Environment


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affinity between the ZIF-8@BHb hybrid composites and TMB was higher than that between free BHb and TMB. In addition, the Vm of the ZIF-8@BHb hybrid composites and free BHb were 6.45 min-1 and 1.14 min-1, respectively, confirming that the catalytic rate of the ZIF-8@BHb hybrid composites was higher than free BHb. Besides, the pseudo-first-order kinetics in connection with TMB could be adopted for our experimental system. As presented in Figure 4B, the intrinsic activity of the ZIF-8@BHb hybrid composites was calculated to be 270 U/mg, approximately 423% higher than free BHb in solution, where the activity of free BHb was 63.8 U/mg. The great enhancement in the catalytic activity of BHb embedded in ZIF-8 in comparison of free BHb was attributed to the stabilization of the hybrid composites and the unique structure of ZIF-8, such as high surface area, porosity and nanoconfinement, which resulted in high accessibility of the substrate to the active sites of the ZIF-8@BHb hybrid composites.

Visual Detection of H2O2 and Phenol. Encouraged by the above results, a facile colorimetric platform involving the ZIF-8@BHb hybrid composites, co-oxidation of phenol and AAP was designed and applied for the detection of H2O2. Certainly, this colorimetric platform was also fitted for phenol in the presence of H2O2 based upon the same principle. Herein, H2O2 with different concentrations was added to PBS solution (0.1 M, pH 7.4), which contained the ZIF-8@BHb hybrid composites (or free BHb), excess phenol and AAP. As shown in Figure 5A, the absorption intensity at 505 nm increased gradually with the increase of H2O2 concentration, and the good linear relationship between the absorbance (A505) and H2O2 concentrations 14 ACS Paragon Plus Environment

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from 0 to 800 µM was achieved and the corresponding correlation coefficient (R) was 0.9975 (Figure 5B). Accordingly, it was clearly observed by naked-eye visualization that the color of the solution was gradually varied from colorless to pale yellow, to pale pink, and to red when the concentration of H2O2 increased (Figure 5C). The result obtained by the naked eye was well in accordance with that of UV-Vis spectrometer, indicating that the developed visual method is feasible. Significantly, the high catalytic activity of the ZIF-8@BHb hybrid composites made it possible for the fast oxidation reaction, which completed this reaction within 4 min. In addition, the limit of detection (LOD) of H2O2 by the naked-eye visualization was lowed to 1µM, which was much better than that of the earlier reported colorimetric H2O2 sensor.47 It even could bear comparison with the result described by Lyu et al.,39 who used Cyt C-MOFs as biocatalyst, but the LOD of H2O2 was 3 nM obtained by fluorescence spectroscopy and ~10 µM by the naked-eye visualization. It was worth noting that the LOD obtained in current work meets the demand of clinical diagnosis (threshold value for cell damage: ~50 µM).48 In comparison with the ZIF-8@BHb hybrid composites, free BHb only afforded a relatively narrow linear ranges of 0-300 µM (R=0.9969), although the same LOD was gained by the naked-eye observation (Figure S4, Supporting Information). Moreover, more than 15 min were required to complete the oxidation reaction, which was much longer than that of the ZIF-8@BHb hybrid composites. The results indicated that the proposed ZIF-8@BHb-based colorimetric approach was highly sensitive and efficient. Such high sensitivity and fast reaction kinetics was mainly attributed to of BHb embedded in ZIF-8. Identically, 15 ACS Paragon Plus Environment


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the quantitative detection of phenol was also performed by the naked-eye visualization and the result was presented in Figure 6, a linear relationship between A505 and phenol concentrations in the ranges of 0-200 µM (R=0.9985) was obtained with a rapid color response (~4 min) by using the ZIF-8@BHb hybrid composites, and the LOD of phenol was approximately 1 µM, which was better than that described in the previous report.34 In comparison, free BHb-based colorimetric system (Figure S5,

Supporting

Information)

showed

an

insensitive

colour

change

and the reaction required a long reaction time (~15 min), although the same linear ranges of 0-200 µM (R=0.9979) and LOD were obtained by the naked-eye observation. The above results validated again the outstanding catalytic activity of the ZIF-8@BHb hybrid composites compared with free BHb in solution, and could be explained by the fact that the low catalytic activity of free BHb in solution lowered the kinetics of the oxidation reaction, which resulted in slow, insensitive changes in color and relatively narrow linear ranges.

Evaluation of the Selectivity and the Effect of Interfering Substances. The selectivity of the developed method was further evaluated and the results were shown in Figure 7A and B. The existence of control compounds with molar equivalent did not cause significant color changes of the solutions. However, an unambiguous color change from colorless to pink was observed by the addition of the target H2O2 (or phenol), indicating the high selectivity of the developed method. The effect of coexistence substances on the detection of H2O2 or phenol was also examined. As listed in Table S2 and S3 (Supporting Information), some of small 16 ACS Paragon Plus Environment

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biomolecules, such as amino acids and monosaccharides that were often co-existed in human cell sap, could be allowed to be more than 500 µM given the tolerance level of ±10% for H2O2 visualization. Besides, some common anions, cations and metal ions could also be endured in the wide ranges (100~500-fold concentration) in the presence of H2O2. Major tested substances except Co2+ and Ni2+ had little effect on the visual detection of phenol (Table S3, Supporting Information) in the same way. We should note that despite these metal ions of Fe2+, Ni2+ and Co2+ has a bad effect on the colorimetric assay of H2O2 and phenol, these interferences can be eliminated by adding masking agent (e.g. EDTA) before analysis.

Real Sample Analysis. The good performance of the ZIF-8@BHb hybrid composites encouraged us to further explore its application in real-world sample. It is well known that reactive oxygen species such as H2O2 would cause genetic and other damage in human body.49 Therefore, it is significant to detect intracellular H2O2 concentrations for health monitoring. As illustrated in Figure 8A(1-2), no H2O2 was detected in cell sap and colorless solution was observed. In contrast, a distinct color change in the spiked cell sap was observed while adding H2O2 and color gradients were presented with the increase of H2O2 concentrations. The results suggested the practicability of the developed method. Visualization of phenol in sewage was shown in Figure 8B(1-2), in which no color change in sewage sample, but the gradual color changes from colorless to pale yellow, and to pink were clearly found in the spiked sewages by the addition of phenol with different concentrations. The results supported again that the ZIF-8@BHb hybrid composites can be served as a colorimetric sensing 17 ACS Paragon Plus Environment


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platform for sensitive colorimetric determination of H2O2 and phenol. Additionally, the recovery of the developed method was also assessed and high recoveries of 90.5~102.5% and 87.6~106.2% for H2O2 and phenol analysis were obtained, respectively.

Reusability and stability. The reusability of the ZIF-8@BHb hybrid composites was tested. As shown in Figure 9, there was less than 1% catalytic activity loss observed for the detection of H2O2 and phenol after successive 10 cycles, demonstrating the excellent reusability of the hybrid composites. In addition, the stability of the ZIF-8@BHb hybrid composites was studied after storing at -20 ℃ for 30 days and the result (data not shown) showed that the catalytic activity of the ZIF-8@BHb hybrid composites more than 96% was retained, confirming the good stability of the hybrid composites.

General applicability of Synthesis of ZIF-8@hemeprotein Hybrid Composites. To evaluate the general applicability of the synthetic method, other hemeproteins, such as HRP and Mb, were selected as the model organic components and the synthetic procedures were the same as that of the ZIF-8@BHb hybrid composites. As shown in Figure S6 (Supporting Information), both of ZIF-8@HRP and ZIF-8@Mb hybrid composites showed some amorphous sheet on surface of the ZIF-8 crystals, demonstrating the successful fabrication of the ZIF-8@hemeprotein hybrid composites. The corresponding catalytic activity of the ZIF-8@HRP and ZIF-8@Mb hybrid composites were enhanced to 291% and 273% compared with free HRP and Mb, respectively (Figure S7, Supporting Information), confirming the 18 ACS Paragon Plus Environment

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excellent catalytic activity of the ZIF-8@hemeprotein hybrid composites.

CONCLUSIONS In summary, we developed a facile one-pot approach for the synthesis of the ZIF-8@BHb hybrid composites. The synthetic method was very simple and efficient, which was also suitable for the preparation of other ZIF-8@hemeprotein hybrid composites. The resultant ZIF-8@BHb hybrid composites exhibited 423% enhancement in catalytic activity in comparison with free BHb in solution. This good performance of the ZIF-8@BHb hybrid composites made it possible to serve as a colorimetric sensing platform for the colorimetric detection of H2O2 and phenol. The developed

method

exhibited

a

high

selectivity,

good

sensitivity,

and

rapid response time towards the target compounds. The successful real-sample applications demonstrate its great potential of the ZIF-8@BHb hybrid composites in biological and environmental analysis. ACKNOWLEDGEMENT This work was financially supported by the National Natural Science Foundation of China (21375018 and 21675025), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT15R11), and the Natural Science Foundation of Fujian Province (2014J01402). ASSOCIATED CONTENT Supporting Information Experimental details, additional images and Tables as presented in text. This information is available free of charge via the Internet at http://pubs.acs.org. 19 ACS Paragon Plus Environment


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Captions Figure 1. (A) Schematic representation of the synthesis of ZIF-8@BHb composites; (B) milky ZIF-8 solution after ultrasound for 10min; (C) SEM images of the ZIF-8@BHb hybrid composites obtained after incubation for 10h. Figure 2. (A1-2) SEM images of ZIF-8; (A3-4) TEM images of ZIF-8; (B1-2) SEM images of ZIF-8@BHb composites; (B3-4) TEM images of ZIF-8@BHb composites. Figure 3. (A) FT-IR spectra of the pure BHb, ZIF-8 and ZIF-8@BHb hybrid composites; (B) TGA curves of ZIF-8 and ZIF-8@BHb hybrid composites; (C) XRD patterns of ZIF-8 and ZIF-8@BHb hybrid composites before (a, b) and after heating (c, d) at 400 ℃; (D) SEM images of ZIF-8 (D1-2) and ZIF-8@BHb hybrid composites (D3-4) treated at 400 ℃. Figure 4. (A) UV-Vis spectra of different reagent in catalytic system; (B) Catalytic kinetics and reaction rate (insert part) of the oxidization of TMB by free BHb and ZIF-8@BHb hybrid composites; Experimental conditions: 37℃ ℃, 1mM TMB, and 3mM H2O2 in sodium



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citrate buffer solution (0.2 M, pH 4.0) for each system. Figure 5. (A) UV-Vis absorption spectra of peroxidase-like oxidation reaction of H2O2 catalyzed by ZIF-8@BHb hybrid composites; (B) plot of A505 versus H2O2 concentration; (C) Colorimetric change of the solutions upon the addition of variable H2O2 concentrations (0-800µM). Figure 6. (A)UV-Vis absorption spectra of peroxidase-like oxidation reaction of phenol catalyzed by ZIF-8@BHb hybrid composites; (B) plot of A505 versus phenol concentration; (C) Colorimetric change of the solutions upon the addition of variable phenol concentrations (0-200µM). Figure 7. (A)Value of A505 and photographs (inset) of the solutions containing (A) H2O2, (B) phenol or various species. Concentration of each test substances: 50 µM. Figure 8. (A1-2) UV-Vis absorption spectra of the cell sap spiked with different concentrations of H2O2 (0, 1, 10 and 100 µM); visual color change of the cell sap. (B1-2) UV-Vis absorption spectra of sewage spiked with different concentrations of phenol (0, 1, 10 and 100 µM); visual color change of the sewage. Figure 9. Reusability of the ZIF-8@BHb hybrid composites in the cycle analysis of H2O2 and phenol. Concentration of each test substances: 50 µM.

Figure 1


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Figure 3


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For TOC only


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