Fe-N-C Single-Atom Nanozyme for the Intracellular Hydrogen

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Fe-N-C Single-Atom Nanozyme for the Intracellular Hydrogen Peroxide Detection Lei Jiao, Weiqing Xu, Hongye Yan, Yu Wu, Chunrong Liu, Dan Du, Yuehe Lin, and Chengzhou Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02901 • Publication Date (Web): 22 Aug 2019 Downloaded from pubs.acs.org on August 22, 2019

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Analytical Chemistry

Fe-N-C Single-Atom Nanozyme for the Intracellular Hydrogen Peroxide Detection Lei Jiao,† Weiqing Xu,† Hongye Yan,† Yu Wu,† Chunrong Liu,† Dan Du,‡ Yuehe Lin,‡ Chengzhou Zhu†* †Key

Laboratory of Pesticide and Chemical Biology of Ministry of Education

International Joint Research Center for Intelligent Biosensing Technology and Health College of Chemistry, Central China Normal University, Wuhan, 430079, P. R. China ‡School

of Mechanical and Materials Engineering, Washington State University, Pullman, Washington 99164, United States

ABSTRACT: Recently, in situ detection of hydrogen peroxide (H2O2) generated from live cells have caused tremendous attention, because it is of great significance in the control of multiple biological processes. Herein, Fe-N-C single-atom nanozymes (Fe-N-C SAzymes) with intrinsic peroxidase-like activity was successfully prepared via high-temperature calcination using FeCl2, glucose, and dicyandiamide as precursors. The Fe-N-C SAzymes with FeNx as active sites were similar to natural metalloproteases, which can specifically enhance the peroxidase-like activity rather than oxidase-like activity. Accordingly, owing to the excellent catalytic efficiency of the Fe-N-C SAzyme, colorimetric biosensing of H2O2 in vitro was performed via a typical 3,3’,5,5’-tetramethylbenzidine induced an allochroic reaction, demonstrating the satisfactory specificity and sensitivity. With regard to the practical application, in situ detection of H2O2 generated from the Hela cells by the Fe-N-C SAzymes was also performed, which can expand the applications of the newborn SAzymes.

Hydrogen peroxide (H2O2), an essential representative of reactive oxygen species, is produced from oxygen metabolism in cells, which plays a vital role in stimulating cellular proliferation, differentiation, and migration.1-3 H2O2 also can be regarded as the biomarkers for reflecting the Parkinson’s disease and Alzheimer disease due to its damage for nucleic acids, proteins, brain, and tissues.4-6 Hence, it is a real need to develop an ultrasensitive and selective approach for detection of H2O2 in living cells. Recently, some advanced methods including electrochemistry, fluorescence,7-9 electrochemiluminescence,10-12 and colorimetric assay,13-15 have been wildly used for H2O2 detection. Among them, by taking advantage of rapid visual detection and no-requirements of any sophisticated instrumentation or expert skills, colorimetric assay exhibits great potential for point-of-care testing.16 Nanozymes are the nanomaterials with enzyme-like activity, which have attracted enormous attention in recent years for the applications of biosensing, biotherapy and environmental protection due to their unique advantages covering high activity and stability, low cost, easy scaled-up and modification.17-19 Since the first attempt of Fe3O4 nanomaterials with intrinsic peroxidase-mimicking activity in 2007,20 more than dozens of nanozymes including carbon nanomaterials,21, 22 transition metal nanomaterials,23-25 and noble metal nanomaterials,26, 27 have been discovered in the last decade. Although some breakthroughs of nanozymes having been achieved, it still remained some challenges including the uncertain catalytic mechanism, low activity and

selectivity. It is a high desire to rationally design and develop novel nanozymes with definite active centers for the understanding of structure-property relationships. Recently, single-atom catalysts (SACs), defined by Zhang and co-workers,28 have made great improvements in electrocatalysis,29-33 and photocatalysis.34 Metallic oxides/sulfides/hydroxides, metal-organic frames and carbon materials have been successfully used as supports for anchoring of metal single atoms.28, 31, 35-37 Among of them, M-N-C SACs with atomically MNx sites were similar to natural metalloenzymes.38 M-N-C SACs are expected to possess enzyme-like activity. Accordingly, the single-atom nanozymes (SAzymes) were reported previously.39, 40 Like traditional nanozymes, SAzymes with multienzyme activities including peroxidase, oxidase, catalase, superoxide dismutase have been used for biosensing, therapy and organic pollutants degradation.40-43 However, intracellular H2O2 detection by the SAzymes has never been reported. Herein, the Fe-N-C SAzyme with metal loading of 1.3 wt% was synthesized by one-pot high-temperature calcination. Using glucose and dicyandiamide (DICY) as precursors can avoid the aggregation of Fe atoms through the interactions between Fe2+ and rich oxygen species in glucose, which can avoid the additional acid-etching. The resultant Fe-N-C SAzyme with intrinsic peroxidase-like activity can catalyze H2O2 to produce •OH at FeNx active sites, similar to that of natural metalloprotease. In addition, the obtained Fe-N-C SAzyme demonstrated high catalytic efficiencies, which were successfully applied for the ultrasensitive and specific

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detection of H2O2 via a typical colorimetric assay. Importantly, as a proof-of-concept application, in situ detection of H2O2 produced from the Hela cells was performed, which paves a new way to expand the applications of Fe-N-C SAzymes (Scheme 1).

Scheme 1. Mechanism of H2O2 detection released from Hela cells by Fe-N-C SAzyme.

EXPERIMENTAL SECTIONS Materials and Instruments. Glucose, dicyandiamide (DICY), 3,3',5,5'-tetramethylbenzidine (TMB), horseradish peroxidase (HRP), 5,5-dimethyl-1-pyrroline-oxide (DMPO) and phorbol-12-myristate-13-acetate (PMA) were obtained from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Terephthalic acid (TA), acetic acid (HAC), sodium acetate (NaAC), ferrous chloride tetrahydrate (FeCl2•4H2O) and hydrogen peroxide (H2O2) were purchased from Singpharm Chemical Reagent Co. Ltd. The transmission electron microscope (TEM) images were measured by a Titan G260-300. X-ray photoelectron spectroscopy (XPS) measurements were performed by Thermo ESCALAB 250XI (Thermo Fisher, United States). X-ray diffraction (XRD) characterization was carried out by a D8 ADVANCE (Bruker, Germany). All enzyme kinetics data and UV-vis spectra were performed by a multimode reader (Tecan Spark, Switzerland). The electron spin resonance spectra were performed by a A300 (Bruker, Germany). The aberration‐corrected high‐angle annular dark‐field scanning transmission electron microscopy (AC-HAADF‐STEM) were performed by a Titan G2-600 (FEI, Unites States). The X-ray absorption fine structure spectra (Fe K-edge) were collected at 1W1B station in Beijing Synchrotron Radiation Facility (BSRF). Preparation of Fe-N-C SAzyme. Glucose (1 g), DICY (5 g), and FeCl2•4H2O (5 mL, 10 mM) were dissolved in water to obtain a homogeneous solution and stirred 12 hours. Next, the water was removed by freeze-drying to obtain the powder. Finally, the powder was pyrolyzed at 900 ℃ for 2 h with a rate of 3 ℃/min in N2 atmosphere. N-C was also prepared in the absence of FeCl2•4H2O as control. Peroxidase Activity and Kinetics Assay of Fe-N-C SAzyme. Kinetic measurements of peroxidase-like property was confirmed by the color change of TMB, which is indicated by the absorbance at 652 nm recorded by a multimode reader. The kinetic data were detected by the change in concentration

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of TMB and H2O2. As for the kinetic data towards TMB, 10 µL of Fe-N-C SAzyme were added into the mixture including 100 µL of H2O2 (100 mM), 150 µL of HAc-NaAc (0.1 M, pH 3.0) and different concentrations of TMB (50 µL). Similarly, as for the kinetic data towards H2O2, 10 µL of Fe-N-C SAzyme were added into the mixture including 50 µL of TMB (1 mM), 150 µL of HAc-NaAc (0.1 M, pH 3.0) and different concentrations of H2O2 (100 µL). The kinetic data were calculated by a typical Michaelis–Menten curve as to v= Vmax[S]/(Km+[S]), where v is the initial velocity, [S] is the concentration of the substrate, Km is the Michaelis−Menten constant, and Vmax is the maximal reaction velocity. Colorimetric Biosensing of H2O2. Various concentrations of H2O2 (100 µL) and some interfering substrates including NaCl, KCl, MgCl2, glucose, and bovine serum albumin (BSA) were added into the 96-microwell plates in the presence of Fe-N-C SAzyme (0.5 mg/mL, 10 µL). Next, TMB (100 µL, 1 mM) and HAC-NaAC buffer solution (100 µL, pH 3.0) were added subsequently for incubation 5 minutes. Finally, the absorbances at 652 nm were recorded by a multimode reader. Cell Culture and Detection H2O2 Produced from the Hela Cells. The Dulbecco’s modified Eagle’s medium were used to culture the Hell cells under oxidative stress conditions supplemented with fetal bovine serum (10%), and then maintained in a humidified atmosphere of 5% CO2 at 37 °C. To evaluated the H2O2 released from Hell cells, Hela cells were dropped into the 96-microwell plates for 24 hours. After that, the plates were washed three times using PBS solution. Then, the PMA solutions (20 µL, 2 µM) and 100 µL of PBS were added successively and incubated 30 minutes. Finally, Fe-N-C SAzyme (0.5 mg/mL, 10 µL), TMB (100 µL, 1 mM) and HAC-NaAC buffer solution (100 µL, pH 3.0) were added subsequently for incubation 5 minutes. Finally, the absorbances at 652 nm were recorded by a multimode reader.

RESULTS AND DISCUSSIONS


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Analytical Chemistry Figure 1. (A) Schematic illustration of the preparation of Fe-N-C SAzyme. TEM image (B), SAED pattern (C) and HRTEM image (D) of the obtained Fe-N-C SAzyme. HAADF-STEM image (E), and HAADF-STEM mapping images of C, N, O, Fe, and merged image (F-J), respectively.

In order to obtain the atomically dispersed Fe-N-C SAzyme, FeCl2, glucose and DICY as the metal precursor and source of carbon and nitrogen. First, glucose with abundant oxygen species can chelate Fe2+ to effectively avoid the aggregation of Fe2+ because of the strong interaction between oxygen-containing groups and metal irons,44 which can efficiently avoid the additional acid-etching in the final step. Next, during the pyrolysis, the cheated complex (Fe2+-O) was decomposed at low temperature (around 500 ℃), where the Fe atoms were simultaneously trapped in the generated CNx through the strong chemical coupling with nitrogen lone-pair electrons.45 Finally, the atomically dispersed Fe-N-C SAzyme was obtained by pyrolysis at 900 ℃ for 2 hours (Figure 1A). The morphology of the Fe-N-C SAzyme was first characterized by transmission electron microscope (TEM). As shown in Figure 1B, the typical graphene nanosheets with drapes were observed. The selected area electron diffraction (SAED) shown in Figure 1C with ring-like features indicated the poor crystallinity of Fe-N-C SAzyme. The high-resolution TEM (HRTEM) image revealed that there are no metal nanoparticles in Fe-N-C SAzyme (Figure 1D). As can be observed from the X-ray diffraction (XRD) patterns in Figure S1A, N-C and Fe-N-C had two broad diffraction peaks, which were fitted well with the (002) and (101) crystal of graphite carbon. No peak corresponding to the Fe-based nanoparticles was detected, demonstrating the nanoparticle-free feature of Fe-N-C SAzyme. The elements of C, N, O, Fe in Fe-N-C SAzyme were detected by the X‐ray photoelectron spectroscopy (XPS) (Figure S1B). High-resolution N 1s analysis revealed that the existence of pyridinic N, FeNx species, pyrrolic N, graphitic N, and oxidized N (Figure S1C). In addition, the high-resolution of Fe 2p had two peaks at 711 and 723 eV (Figure S1D), which could prove the existence of oxidized Fe species in Fe-N-C SAzyme. High‐angle annular dark‐field scanning transmission electron microscopy (HAADF‐STEM) (Figure 1E) and energy‐dispersive X‐ray spectroscopy (EDS) mapping (Figure 1F-J) indicate that C, N, O, and Fe elements are distributed uniformly within Fe-N-C SAzyme. The Fe atom loading was confirmed as to 1.3 wt% by the inductively coupled plasma optical emission spectrometer.

Figure 2. (A-B) AC-HAADF-STEM images of Fe-N-C SAzyme. Normalized XANES spectra at Fe K-edge of the Fe foil, Fe2O3 and Fe-N-C SAzyme (C), and the corresponding (D) k3-weighted Fourier transform spectra.

The atomically dispersed Fe species in Fe-N-C SAzyme was further verified by the aberration‐corrected HAADF‐STEM (AC-HAADF‐STEM). As exhibited in Figure 2A-B, abundant isolated bright dots were uniformly dispersed on the surface of Fe-N-C SAzyme, which were attributed to the Fe single atoms. Furthermore, the electronic and structural information of Fe species in Fe-N-C SAzyme were investigated by X‐ray absorption near‐edge structure (XANES) and extended X‐ray absorption fine structure (EXAFS) spectroscopy at the Fe K‐edge. As for XANES spectra (Figure 2C), the near‐edge absorption energy of Fe-N-C SAzyme located between the Fe foil and Fe2O3, demonstrating the positively charged Fe single atoms in Fe-N-C SAzyme. Moreover, the EXAFS curve of Fe-N-C SAzyme showed a primary peak at about 1.5 Å, which was assigned to the Fe-N scattering paths, and the Fe-Fe peak at 2.2 Å was not detected (Figure 2D). According to the EXAFS fitting parameters (Table S1), an average Fe-N coordination number of 4.3 was obtained. As a consequence, according to the AC-HAADF‐STEM images, XANES and EXAFS analysis, the existence of single Fe atoms in Fe-N-C SAzyme can be confirmed undoubtedly.



generation of free •OH by using 5,5-dimethyl-1-pyrroline-oxide (DMPO) as the specific probe, agreed well with the fluorescent signal mentioned above. Hence, the Fe-N-C can catalyze H2O2 to produce •OH as active intermediates, which can oxidize TMB to produce the color changes. To quantificationally evaluate the peroxidase-like activity of Fe-N-C SAzyme, the Michaelis– Menten constant (KM) and maximum velocity (Vmax) were performed subsequently (Figure S3A-B). According to the double reciprocal plots of initial reaction rates, the enzyme kinetic parameters are summarized in Table S2. In comparison to other reported nanozymes with peroxidase-like activity,46, 47 the newly-designed Fe-N-C SAzyme exhibited the highest Kcat/Km, demonstrating the best peroxidase-like activity (Table S2), which can be attributed to the maximum atomic utilization, and unsaturated coordination environment of Fe-N-C SAzyme.

Figure 3. (A) Active sites of HRP and Fe-N-C SAzyme. (B) UV-vis absorption spectra of TMB, H2O2+TMB, Fe-N-C SAzyme+H2O2+TMB. (C) Comparison of oxidase and peroxidase activities of N-C nanozyme and Fe-N-C SAzyme. (D) Fluorescent curves of TA, H2O2+TA, N-C+H2O2+TA, and Fe-N-C SAzyme+H2O2+TA. (E) ESR spectra of H2O2+DMPO, N-C+H2O2+DMPO and Fe-N-C SAzyme+H2O2+DMPO.

Due to the similar FeNx active sites to the natural horseradish peroxidase (HRP), Fe-N-C SAzymes with homogeneous FeNx active sites are expected to possess the intrinsic peroxidase activity (Figure 3A). As expected, the Fe-N-C SAzyme exhibited excellent peroxidase activity verified by a typically chromogenic reaction by the catalytic oxidation of 3,3′,5,5′‐tetramethylbenzidine (TMB) with the help of H2O2. The Fe-N-C SAzyme can rapidly oxidize TMB and produce an obvious color change at 652 nm with the help of H2O2, whereas individual TMB, and TMB/H2O2 system could not trigger the color changes (Figure 3B). Furthermore, we compared the oxidase- and peroxidase-like activities of N-C nanozyme and Fe-N-C SAzyme. Although the oxidase-like activity of Fe-N-C SAzyme was higher than that of N-C nanozyme, the enhancement of oxidase-like activity was far less than that of peroxidase-like activity (Figure 3C). The Fe single atoms involved in Fe-N-C SAzyme can specifically enhance the peroxidase-like activity rather than oxidase-like activity. Similar to natural HRP, the peroxidase activity of Fe-N-C SAzyme was also high concentration- and pH-dependent (Figure S2A-B). As displayed in Figure S2C, the catalytic activity of natural HRP was declined with the increment of incubation temperature. Note that Fe-N-C SAzyme can maintain considerable catalytic efficiency even treated at 80 ℃. After five cycles, the catalytic activity of the Fe-N-C SAzyme still remained unchanged (Figure S2D). Furthermore, we evaluated the reactive intermediates during the H2O2 catalysis by the Fe-N-C SAzyme. Terephthalic acid (TA) as a typically fluorescent probe can capture hydroxyl radical (•OH) to produce a fluorescent product with an emission at 440 nm. As expected, only if the Fe-N-C SAzyme, H2O2, and TA are all existed, an obvious fluorescent signal was observed, demonstrating the presence of •OH during H2O2 catalysis by the Fe-N-C SAzyme (Figure 3C). Fe-N-C SAzyme exhibited higher fluorescent intensity than N-C. The electron spin resonance (ESR) results also indicated the

Figure 4. UV-vis absorption spectra of TMB oxidized by the Fe-N-C SAzyme with different concentrations of H2O2 (A) and its corresponding calibration curve (B). The reproducibility (C) and selectivity (D) of H2O2 detection by the Fe-N-C SAzyme.

By taking advantage of the outstanding peroxidase-like activity of Fe-N-C SAzyme, the colorimetric biosensing of H2O2 was further performed in Figure 4. As shown in Figure 4A, following the increment of H2O2 concentration, the absorbance values generated by the TMB oxidation were gradually increased. Accordingly, a fine linear relationship curve between H2O2 concentration and absorbance values was achieved in the range from 0.5 to 100 mM (Figure 4B). In addition, the reproducibility and selectivity of the H2O2 detection by Fe-N-C SAzyme were evaluated. As displayed in Figure 4C, after five repeated measurements, the absorbances of oxidized TMB still kept the same as original values in 100 mM H2O2, indicating the acceptable reproducibility of Fe-N-C SAzyme. Furthermore, to evaluate the selectivity of H2O2 detection, various interfering substrates were used to study the specificity of H2O2 detection by Fe-N-C SAzyme. Compared with 100 mM H2O2, the Zn2+, Mn2+, Ca2+, glucose, BSA, glutathione (GSH), dopamine (DA), L-cysteine (L-Cys), and ascorbic acid (AA) with the concentration of 500 mM did not cause obvious color changes, indicating the satisfactory selectivity of H2O2 detection by Fe-N-C SAzyme (Figure 4D).


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Analytical Chemistry

ASSOCIATED CONTENT Supporting Information XRD and XPS of Fe-N-C SAzyme; Optimizing concentration, pH, temperature of Fe-N-C SAzyme; Dynamics data of Fe-N-C SAzyme.

AUTHOR INFORMATION Corresponding Author * Chengzhou Zhu, E-mail: [email protected]

ORCID Chunrong Liu: 0000-0002-9240-0380 Dan Du: 0000-0003-1952-4042 Yuehe Lin: 0000-0003-3791-7587 Chengzhou Zhu: 0000-0003-0679-7965 Figure 5. MTT assay of the Fe-N-C SAzyme (A) and TMB (B). (C) Absorbance values of the Hela cells treated with different concentrations of PMA. (D) Absorbance values of the different number of Hela cells treated with or without PMA.

For practical applications, determination of H2O2 generated from the Hela cells was performed. First, the potential toxicity of Fe-N-C SAzyme was verified by the standard MTT assay. The Hela cells can maintain considerable viability even when the Hela cells were incubated with a high concentration (50 µg/mL) of Fe-N-C SAzyme, testifying the good biocompatibility of Fe-N-C SAzyme (Figure 5A). The added TMB had little effects on cell viability (Figure 5B). Then, in situ detection of H2O2 produced from Hela cells was carried out. In detail, PMA as H2O2 inducers were used to stimulate Hela cells. The generated H2O2 were captured by Fe-N-C SAzyme to produced •OH, which is responsible for the TMB oxidation to produce an obvious color change at 652 nm. As shown in Figure 5C, the Hela cells were treated with PMA with different concentrations, demonstrating a high concentration-dependent feature. In addition, the Hela cells with different cell numbers were treated with or without PMA. Following the increment of cell numbers, a higher colorimetric response was observed, which can be attributed to the more H2O2 produced in the PMA stimulation (Figure 5D). Furthermore, according to the calibration curve of H2O2 detection in Figure 4B, the concentration of H2O2 produced from the Hela cells (1106 cells/plate) was calculated to be 1.99 mM. Hence, based on the Avogadro's constant (NA = 6.02×1023 mol−1), the average number of H2O2 released one cell (N0) was to 3.951011 (calculated by the Avogadro equation: n=N0/NA), which was consistent with reported works previously.48, 49

CONCLUSION To sum up, the Fe-N-C SAzyme with atomically dispersed Fe-N center sites was successfully prepared via high-temperature calcination, exhibiting outstanding peroxidase-like activity. The atomically dispersed Fe-N center sites were similar to natural HRP, which can specifically enhance the peroxidase-like activity instead of oxidase-like activity. Accordingly, the resultant Fe-N-C SAzyme exhibited satisfactory sensitivity and specificity for colorimetric biosensing of H2O2 in vitro. In addition, in situ detection of H2O2 generated from the Hela cells was performed, which could expand the applications of Fe-N-C SAzyme in intracellular biosensing.

Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT The authors gratefully acknowledges the financially supported by the self-determined research funds of CCNU from the colleges' basic research and operation of MOE (CCNU18JCXK07), Fundamental Research Funds for the Central Universities (CCNU19QN066), the Program of Introducing Talents of Discipline to Universities of China (111 program, B17019) and the Recruitment Program of Global Youth Experts of China.

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