Highly selective generation of singlet oxygen from dioxygen with atomically dispersed catalysts

Singlet oxygen (1O2) as an excited electronic state of O2 plays a significant role in ubiquitous oxidative processes from enzymatic oxidative metabolism to industrial catalytic oxidation. Generally, 1O2 can be produced through thermal reactions or the photosensitization process; however, highly selective generation of 1O2 from O2 without photosensitization has never been reported. Here, we find that single-atom catalysts (SACs) with atomically dispersed MN4 sites on hollow N-doped carbon (M1/HNC SACs, M = Fe, Co, Cu, Ni) can selectively activate O2 into 1O2 without photosensitization, of which the Fe1/HNC SAC shows an ultrahigh single-site kinetic value of 3.30 × 1010 min−1 mol−1, representing top-level catalytic activity among known catalysts. Theoretical calculations suggest that different charge transfer from MN4 sites to chemisorbed O2 leads to the spin-flip process and spin reduction of O2 with different degrees. The superior capacity for highly selective 1O2 generation enables the Fe1/HNC SAC as an efficient non-radiative therapeutic agent for in vivo inhibition of tumor cell proliferation.


Introduction
Dioxygen (O 2 ) occupies a critical position in a great variety of oxidation reactions involved in both chemical reactions and biological processes. [1][2][3][4][5][6] In aerobic biology, O 2 -related oxidation reactions are achieved by a series of evolutionary metalloenzymes such as cytochrome P450 through the interaction with O 2 , resulting in the formation of reactive oxygen species (ROS) such as singlet oxygen ( 1 O 2 ), hydroxyl radical (cOH) and superoxide anion (O 2 À ) or reactive metal-O 2 intermediates including metal superoxo, (hydro)peroxo, and metal-oxo species. [7][8][9][10][11] Among the active species, 1 O 2 with an unoccupied p* orbital has strong electrophilicity and unique reactivity and selectivity to serve as a synthetic reagent, attracting enormous interest from both fundamental studies and practical application elds. [12][13][14][15][16] To this end, increasing attention has been drawn to the development of efficient approaches to highly selective generation of 1 O 2 . [17][18][19][20][21][22] Conventional approaches to 1 O 2 production mainly include thermal processes using enzymatic or chemical reactions and photosensitization of O 2 with rationally designed photosensitizers. [23][24][25][26] However, the production of 1 O 2 through these approaches is always accompanied by other ROS generation. In addition, thermal processes oen require harsh conditions such as rigorous pH and particular solvents. 27,28 Despite the simplicity and controllability of the photosensitization route, it faces inherent limitations like poor selectivity of 1 O 2 , photobleaching of sensitizers, and difficulty in large-scale production. [29][30][31] Therefore, development of new approaches to highly selective production of 1 O 2 is of great signicance not only in the investigation of 1 O 2 -related biological and physical processes but also in the development of new materials and biological tools with 1 Recently, single-atom catalysts (SACs) have shown considerable potential in industrial chemical processing and photo/ electrochemical energy conversion with high catalytic activity and unique selectivity due to their excellent properties like maximum atom utilization efficiency, well-dened active centers, and tunable coordination environment. [32][33][34] Among the developed SACs, metal-nitrogen (MN x ) sites embedded in carbon skeletons represent a signicant series of SACs with excellent catalytic activity toward oxygen-related reactions including the oxygen reduction reaction and advanced oxidation processes, benetting from precise tuning of electronic structures of active sites. [35][36][37][38] Thus, MN x SACs provide great possibilities and opportunities in the design and fabrication of selective catalysts for 1 O 2 production. However, despite some investigations on the capacity of MN x SACs toward oxygen activation with non-selective ROS production, the atomic engineering of MN x SACs is still needed to optimize the metal-O 2 interaction to achieve selective 1 O 2 generation.
Here, we demonstrate a new approach to highly selective generation of 1 O 2 from O 2 without photosensitization with single transition metal atoms anchored on hollow N-doped carbon as atomically dispersed catalysts (M 1 /HNC SACs, M ¼ Fe, Co, Cu, Ni). The 1 O 2 generation efficiency is highly related to the metal centers, following the sequence of Fe 1 /HNC > Co 1 / HNC > Cu 1 /HNC > Ni 1 /HNC. Among the catalysts examined, the Fe 1 /HNC SAC with a single atomic motif of FeN 4 coordination shows the best kinetic value of 0.140 min À1 , exceeding those of Co 1 /HNC (0.033 min À1 ), Cu 1 /HNC (0.019 min À1 ) and Ni 1 /HNC (0.016 min À1 ), and the single-site kinetic value of the Fe 1 /HNC SAC reaches up to as high as 3.30 Â 10 10 min À1 mol À1 , representing top-level catalytic activity among known catalysts. Density functional theory (DFT) calculations demonstrate that the selective 1 O 2 generation capacity originates from signicant charge transfer from MN 4 sites to chemisorbed O 2 , leading to the spin-ip process and spin reduction of O 2 with the lowest value of 0.25 for O 2 adsorbed on the FeN 4 site. Based on the efficient production of 1 O 2 enabled by the Fe 1 /HNC SAC, we develop a non-radiative therapeutic platform for in vivo inhibition of tumor cell proliferation.

Results and discussion
To synthesize M 1 /HNC SACs, metal acetylacetonate encapsulated zeolitic imidazolate framework-8 (M/ZIF-8) was rst synthesized as the raw material to form core-shell composites by tannic acid (TA) coating. The core-shell precursors were then pyrolyzed at 900 C under an Ar atmosphere to obtain M 1 /HNC catalysts (Fig. 1a). Hollow N-doped carbon (HNC) was prepared with pure ZIF-8 without the encapsulation of metal acetylacetonate as the control catalyst. X-ray diffraction (XRD) patterns of the catalysts show peaks for (002) and (101) planes of graphitic carbon, located at around 24 and 43 , respectively, and no observable peaks for metal-related nanoparticles were found ( Fig. S1 †), indicating the absence of metal nanoparticles in M 1 / HNC catalysts. 39 Raman spectra of the M 1 /HNC SACs and HNC reveal similar intensity ratios of the D band to the G band ( gave a similar Brunner-Emmet-Teller (BET) surface area at around 700 cm 2 g À1 , further indicating the structural similarity of the prepared catalysts. Transmission electron microscopy (TEM) and high-angle annular dark-eld scanning TEM (HAADF-STEM) images show that Fe 1 /HNC has a hollow polyhedral morphology with an average size of 200 nm and thickness of 10 nm and no metal nanoparticles were observed (Fig. 1b). The elemental mapping images show a homogeneous distribution of Fe, N, and C elements over the whole domain of the Fe 1 /HNC SAC (Fig. 1c). Furthermore, the aberrationcorrected HAADF-STEM image was obtained to conrm single atomic Fe in the Fe 1 /HNC SAC. As shown in Fig. 1d, the bright dots highlighted with red circles clearly demonstrate that the atomically dispersed Fe atoms exist over N-doped carbon. Similarly, the morphology of other three M 1 /HNC SACs and the atomic dispersion were conrmed by using TEM and HAADF-STEM images, all showing a hollow polyhedral structure and atomically dispersed metal atoms ( Fig. S4-S6 †).
The atomic electronic structure and coordination conguration of metal species in M 1 /HNC SACs were explored by X-ray absorption near-edge structure (XANES) and extended X-ray absorption ne structure (EXAFS) characterization experiments. Fig. 2a shows the XANES spectra of Fe 1 /HNC at the Fe Kedge in comparison with those of references including Fe foil and Fe 2 O 3 . The peak position of Fe 1 /HNC is situated between those of Fe 2 O 3 and Fe foil, indicating that the valence state of the Fe atom is between 0 and +3. The Fourier-transform EXAFS spectrum of Fe 1 /HNC shows only one peak at about 1.5Å, ascribed to the rst coordination shell of Fe-N, and no Fe-Fe peak like that of Fe foil at around 2.2Å was observed (Fig. 2b), suggesting the inexistence of metal-related nanoparticles or clusters in Fe 1 /HNC. In order to further investigate the atomic distribution of Fe in Fe 1 /HNC, wavelet transform (WT) analysis of Fe K-edge EXAFS was carried out. As illustrated in Fig. 2c, only one intensity maximum at approximately 4.2Å À1 related to Fe-N coordination was observed from the WT contour plot of Fe 1 / HNC, which is different from the contour WT plots of Fe foil and Fe 2 O 3 with the intensity maximum corresponding to Fe-Fe contribution, elucidating that no Fe-Fe bond is present in Fe 1 / HNC, but isolated Fe atoms exist. Moreover, the coordination conguration and structural parameters of Fe atoms were obtained from EXAFS tting curves ( Fig. 2d and S7 †). As listed in Table S1, † the coordination number of Fe atoms and the bond length of Fe-N are 4.2 and 2.02Å, respectively. From the above results, the atomic structure conguration of Fe 1 /HNC was constructed (Fig. 2e). The local structure of other three M 1 /HNC SACs was also conrmed with XANES and EXAFS spectra ( Fig. S8-S10 †), all possessing similar atomic conguration with Fe 1 /HNC.
In order to systematically evaluate the oxidizing capacity of M 1 /HNC SACs, the oxidation of colorless 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) to blue oxidized TMB (ox-TMB) with the characteristic adsorption at ca. 652 nm was used as the catalytic model reaction. [40][41][42][43] Considering that the pyrolysis temperature has signicant effect on the coordination microenvironment of SACs, 44,45 the TMB oxidation activities of the catalysts prepared at different temperatures were explored. As shown in Fig. 3a, the catalyst obtained at 900 C (denoted as Fe 1 /HNC-900) exhibits the highest catalytic activity towards TMB oxidation, and 900 C was thus chosen to prepare other SACs. We next compared the catalytic activity of M 1 /HNC SACs with different transition metal centers toward TMB oxidation. The results displayed in Fig. 3b and S11 † show that the activity trends follow the sequence of Fe 1 /HNC > Co 1 /HNC > Cu 1 /HNC > Ni 1 /HNC > HNC, indicating that the single metal atom center plays a vital role in the oxidation reaction. Among the catalysts investigated, Cu 1 [46][47][48] To verify whether O 2 is the only substrate for the oxidation reaction, Fe 1 /HNC was selected as the representative catalyst to evaluate the TMB oxidation reaction under a N 2 atmosphere. As shown in Fig. 3c, the absorbance intensity of ox-TMB undergoes a sharp decrease in the N 2 -saturated B-R buffer compared with that under air-saturated conditions, indicating the indispensable role of O 2 in the oxidation of TMB catalyzed by the Fe 1 /HNC SAC. In addition, as displayed in Fig. S12-S15, † the catalytic  , and 1 O 2 , respectively. 49 Notably, only NaN 3 exhibits efficient inhibitation of TMB oxidation catalyzed by the Fe 1 /HNC SAC, while the others display no obvious effects on the oxidation reaction (Fig. 3d), suggesting that the Fe 1 /HNC SAC selectively activates O 2 into 1 O 2 . In consideration of photosensitization usually implemented to produce 1 O 2 , the comparison of the Fe 1 /HNC-catalyzed TMB oxidation reaction under normal light conditions and in a dark environment was performed. As shown in Fig. 3e, a negligible difference in characteristic absorbance of ox-TMB was observed, indicating the striking generation of 1 O 2 without photosensitization. Collectively, highly selective generation of 1 O 2 from O 2 can be achieved by using the Fe 1 /HNC SAC without the assistance of the externally applied stimulus.
To further validate the selective production of 1 O 2 from O 2 enabled by M 1 /HNC SACs, an 1 O 2 -specic probe 9,10anthracenediyl-bis(methylene) dimalonic acid (ABDA) was adopted to selectively recognize and react with 1 O 2 . 50,51 ABDA is a uorescent molecule with l ex and l em at ca. 380 nm and 433 nm, respectively, which turns to be a non-uorescent endoperoxide product upon selective oxidation by 1 O 2 via a [4 + 2]-cycloaddition. With ABDA as the probe, we evaluated the catalytic efficiency of M 1 /HNC SACs in generating 1 O 2 from O 2 . As can be seen in Fig. 3f and S16, † the activity towards ABDA oxidation follows the rate sequence of Fe 1 /HNC (99.8%) > Co 1 / HNC (42.8%) > Cu 1 /HNC (30.9%) > Ni 1 /HNC (23.4%) > HNC (17.3%), which is consistent with the trends in TMB oxidation. Notably, Fe 1 /HNC displays the highest catalytic activity toward ABDA oxidation with a rate of nearly 100%, indicative of more efficient 1 O 2 generation catalyzed by Fe 1 /HNC in comparison with other SACs. In addition, the kinetic proles of ABDA oxidation were examined to assess 1 O 2 generation capacities of M 1 /HNC SACs by calculating the oxidation rate of ABDA with pseudo-rst-order approximation. As shown in Fig. S17, † an exponential decrease in the uorescence intensity of ABDA against the reaction time was observed and the pseudo-rstorder kinetic constants (k values) were obtained from the linear relationship. The results shown in Fig. 3g reveal that Fe 1 / HNC catalyzes the generation of 1 O 2 for ABDA oxidation with a k value of 0.140 min À1 , considerably higher than that of Co 1 /HNC (0.033 min À1 ), Cu 1 /HNC (0.019 min À1 ) and Ni 1 /HNC (0.016 min À1 ), further highlighting the critical role of different single metal atom coordination. The single-site kinetic constant (k single-site value) of the Fe 1 /HNC SAC was further evaluated to be as high as 3.30 Â 10 10 min À1 mol À1 due to the high catalytic efficiency with a low metal content of 0.17 wt% determined via inductively coupled plasma optical emission spectrometry (ICP-OES). Besides, the Fe 1 /HNC-enabled selective generation of 1 O 2 was investigated with different concentrations of the catalyst and in reaction media with different pH values ( Fig. 3h and S18 †). As shown in Fig. S19, † the ABDA oxidation reaction shows a dose-dependent behavior. In addition, the generation of 1 O 2 for ABDA oxidation is peculiarly prone to occur under acidic conditions.
To further conrm the selective generation of 1 O 2 enabled by Fe 1 /HNC, colorimetric and uorescent probes for other ROSs were utilized, thereinto, r-phthalic acid (PTA) and iodonitrotetrazolium chloride (INT) were selected to detect cOH, and O 2 À , respectively. PTA, as a widely used cOH probe, can be selectively hydroxylated upon reaction with cOH to form 2-hydroxy terephthalic acid with strong uorescence at l ex /l em ¼ 315/ 400 nm. 52 INT is a specic probe for O 2 À , which can be reduced by O 2 À to generate the colored formanzan product with the maximum absorption at 510 nm. As shown in Fig. S20, † no obvious changes in the uorescence intensity of PTA or absorbance of INT were observed in the oxidation reaction, demonstrating negligible generation of cOH and O 2 À but highly selective production of 1 O 2 during the O 2 activation process. Furthermore, electron spin resonance (ESR) characterization using 2,2,6,6-tetramethylpiperidine (TEMP) as the 1 O 2 trapping agent and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) as the trapping agent for cOH and O 2 À were also conducted to give more direct evidence of the produced ROS. 53,54 Fig. 3i shows the typical 1 : 1 : 1 triplet ESR signal for 2,2,6,6tetramethylpiperidine-N-oxyl (TEMPO) formed due to the 1 O 2 generation in the presence of the Fe 1 /HNC SAC. However, there are no observable signals when BMPO was used as the trapping agent (Fig. S21 †), indicating that negligible cOH and O 2 À are produced in the process of Fe 1 /HNC-catalyzed O 2 activation.
To gain in-depth insight into the mechanism of selective 1 O 2 generation enabled by M 1 /HNC SACs, DFT calculations were performed to explore the electronic structure of the active sites and their interactions with O 2 . [55][56][57][58] In accordance with the atomic structural analysis, the models of M 1 /HNC SACs adsorbed with O 2 were constructed and optimized. The adsorption energy of O 2 on the FeN 4 site in the Fe 1 /HNC SAC was calculated to be À1.039 eV, which is less than those of other M 1 /HNC SACs (Fig. 4i), illustrating the strong interaction between the FeN 4 site and O 2 . In order to further elucidate the electronic interactions between MN 4 sites and absorbed O 2 , the projected density of states (PDOS) for M 3d and O 2p was calculated. As shown in Fig. 4a-d, more hybridization between the PDOS for Fe 3d orbitals and O 2p orbitals can be evidently observed in contrast with those for CuN 4 or NiN 4 centers. Meanwhile, the Fe 3d orbitals shi toward the Fermi level upon O 2 adsorption (Fig. S22-S25 †), further demonstrating the occurrence of O 2 activation on FeN 4 . 59,60 In addition, the Bader charge analysis and charge density difference analysis (Fig. 4e-h)  Having demonstrated the high efficiency of selective 1 O 2 generation without photosensitization from molecular O 2 enabled by the Fe 1 /HNC SAC, we then employed the Fe 1 /HNC SAC as a therapeutic agent for suppressing tumor cell growth. To improve the dispersibility and biocompatibility of Fe 1 /HNC, an amphipathic molecule DSPE-PEG2000 was modied on the surface of Fe 1 /HNC to form PEGylated Fe 1 /HNC (donated as P-Fe 1 /HNC) through the hydrophobic interaction. 61 The cellular uptake of P-Fe 1 /HNC was visualized by confocal laser scanning microscopy (CLSM) imaging with uorescein isothiocyanate (FITC)-loaded P-Fe 1 /HNC. As displayed in Fig. 5a and S26, † the bright green uorescence and the black dots in cytosol clearly indicate the efficient internalization of P-Fe 1 /HNC in HeLa cells. Next, the inhibition effect of P-Fe 1 /HNC on cell proliferation was systematically explored by the cell viability test with a standard MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] assay. The results show that the treatment of HeLa cells with PEGylated HNC (P-HNC) without single Fe atomic centers exhibits a negligible effect on the proliferation of HeLa cells with cell viability exceeding 90% even at a concentration of P-HNC as high as 0.3 mg mL À1 (Fig. 5b), indicating the excellent biocompatibility and low cytotoxicity of P-HNC. In striking contrast, an obvious decrease in cell viability of HeLa cells to less than 50% upon the treatment of P-Fe 1 /HNC with a low concentration of 0.06 mg mL À1 was observed and the cell viability decreases with increasing P-Fe 1 /HNC concentration, providing direct evidence for the antiproliferation effect on HeLa cells.
In the meantime, the cytotoxicity of P-Fe 1 /HNC was further investigated by co-staining HeLa cells with calcein-AM (green uorescent dye for viable cell staining) and propidium iodide (PI, red uorescent dye for dead cell staining) and visualized with CLSM imaging. 62 As exhibited in Fig. 5c and S27, † in comparison with the group treated with PBS or P-HNC showing bright green uorescence, cancer cells treated with P-Fe 1 /HNC show strong red uorescence, indicating the high cytotoxicity against HeLa cells of Fe 1 /HNC SACs. According to the results demonstrated above, such capacity of killing cancer cells was deemed to originate from the intracellular oxidative stress induced by Fe 1 /HNC SAC-enabled selective production of 1 O 2 , which was distinctly conrmed with a cell-permeable ROS-sensitive probe (2 0 ,7 0 -dichlorouorescein diacetate, DCFH-DA). 63,64 Obviously, cancer cells treated with P-Fe 1 /HNC show the brightest green uorescence of DCF ( Fig. 5d and S28 †), indicative of most 1 O 2 generation enabled by Fe 1 /HNC. In order to further demonstrate the cytotoxicity against HeLa cells of the Fe 1 /HNC SAC from the oxidative damage induced by the generation of 1 O 2 , ascorbic acid (AA) as an antioxidant was used to pre-incubate HeLa cells. 61 As shown in Fig. 5e, the preincubation of AA can efficiently reduce the cytotoxicity of the Fe 1 /HNC SAC through the elimination of generated 1 O 2 with a concentration-dependent behavior.
The antitumor effect of the Fe 1 /HNC SAC in vivo was also investigated with HeLa tumor-bearing mice as a model. When the tumor volume reached about 100 mm 3 , the mice were randomly divided into three groups and intravenously injected with PBS, P-HNC, and P-Fe 1 /HNC, respectively. As shown in Fig. 5f, the mice treated with P-HNC exhibit a similar tumor growth tendency with that of PBS, indicating that HNC had no obvious therapeutic effect on suppressing tumor growth. In contrast, the group treated with P-Fe 1 /HNC displays a relatively higher tumor inhibition effect with the reduction of the tumor size to 46.5% in 12 days compared with the control group, which was attributed to the high capacity of the Fe 1 /HNC SAC for 1 O 2 generation within the tumor site. Moreover, photographs of the excised tumors further conrmed the good antitumor effect of Fe 1 /HNC (Fig. S29 †). In addition, no obvious changes of the body weight of mice were observed during the experimental period upon administration of different catalysts, indicating the favorable biocompatibility of the Fe 1 /HNC SAC (Fig. 5g). Further blood biochemical analysis also implied the good biosafety of these catalysts (Fig. S30 †).

Conclusions
In summary, we have demonstrated that atomically dispersed MN 4 sites on hollow N-doped carbon can be used for the selective generation of strong oxidizing 1 O 2 from O 2 without photosensitization. The as-prepared M 1 /HNC SACs show different 1 O 2 generation efficiencies in the sequence of Fe 1 /HNC > Co 1 /HNC > Cu 1 /HNC > Ni 1 /HNC. Among the developed catalysts, the Fe 1 /HNC SAC shows the best kinetic value of 0.14 min À1 and a single-site kinetic value of 3.30 Â 10 10 min À1 mol À1 , originating from the spin-ip process and spin reduction of O 2 to 0.25 induced via signicant charge transfer with 0.44 e from the FeN 4 site to O 2 . More importantly, the Fe 1 /HNC SAC with superior 1 O 2 generation capacity has been successfully utilized as an efficient non-radiative therapeutic agent for inhibiting tumor cell proliferation in vitro and in vivo. We believe that this nding would provide a facile method for the selective production of 1 O 2 , opening up a new avenue for the development of functional SACs in biomedical applications.

Data availability
All experimental and computational data is available in the ESI. †

Conflicts of interest
There are no conicts to declare.