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Full-range pH Stable Au-clusters in Nanogel for ConfinementEnhanced Emission and Improved Sulfide Sensing in Living Cells Xilin Bai, Suying Xu, and Leyu Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04785 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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Analytical Chemistry

Full-range pH Stable Au-clusters in Nanogel for ConfinementEnhanced Emission and Improved Sulfide Sensing in Living Cells Xilin Bai†, Suying Xu†, and Leyu Wang* State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, P. R. China. Email: [email protected], Telephone: (86)010-64427869 ABSTRACT: The sensitive and selective detection of hydrogen sulfide is of great importance due to its crucial role in pathological and physiological processes. Herein, we report a novel fluorescent platform, AuNCs@GC, for selective detection of hydrogen sulfide in living cells by impregnating the Au nanoclusters (AuNCs) into a biocompatible cationic polymer matrix, glycolchitosan (GC) nanogel. The confinement effect significantly increased the emissive Au(I) units, resulting in a 6-fold enhancement of quantum yield (from 6.38% to 36.42%). In addition, the prepared positively charged AuNCs@GC nanogel exhibit excellent selectivity and improved sensitivity to aqueous sulfides. Moreover, the as-fabricated AuNCs@GC showed very good biocompatibility and super fluorescence stability across fullrange pH and good salt tolerance, which demonstrated excellent adaptability toward intracellular sulfide imaging.

Hydrogen sulfide (H2S), a member of the reactive sulfur species (RSS) family, which often exists as protonated (H2S), monoanion (HS-) and dianion (S2-) forms in water, plays vital roles in regulating intracellular redox status and other signaling processes,1-4 including vasodilation, oxygen sensing, angiogenesis, inflammation and it can also protect tissues against the damage caused by ischemia or reperfusion.5, 6 In addition, studies have shown that abnormal levels of H2S can be important indicators for many serious diseases, such as Alzheimer’s disease, Down’s syndrome, diabetes and liver cirrhosis.7, 8 Ever-increasing interests in sulfide demand more sensitive and reliable detection techniques,9-12 particularly for reliable in situ detection and imaging. In this regard, fluorescent sensing and imaging is considered as one of the most promising strategies owing to its high sensitivity, good selectivity and real-time merits.13-21 To date, many fluorescent sensors22, 23 was constructed by taking advantage of the strong metalsulfide complexation and/or the reductive properties of sulfides. Yet, real-time intracellular sulfide imaging was still desirable. Metal nanoclusters (NCs, such as Au, Ag, Cu and Pt) are a kind of luminophor with unique molecule-like properties and discrete electronic energy levels.24, 25 Owing to their remarkable optical and chemical properties, these NCs have been widely used in molecular recognition and other biomedical studies.26-28 However, the quantum yields (QYs) of water dispersible luminescent NCs are normally lower than those well-

established chromophores, such as organic dyes and inorganic quantum dots, which greatly limits their optical applications.2933 At this point, extensive efforts have been devoted to fabricate stable and highly luminescent NCs.34 It is widely accepted that the aggregation of Au(I)-thiolate complex accounts for luminescence of gold nanoclusters (AuNCs). Thus, increasing the portion of Au(I)-thiolate complex as well as the aggregation degree has been considered an effective way for the preparation of highly luminescent AuNCs.35 Alternatively, the luminescence could be enhanced by encapsulating AuNCs in a polymer matrix such as chitosan that protected them from unwanted relaxation and rigidified the structure.36, 37 Despite of great progresses in the fabrication of luminescent AuNCs, it is still highly challenging to prepare highly luminescent AuNCs with excellent stability such as good tolerance of wide pH range and salinity conditions. Herein, we report a facile strategy for the fabrication of highly stable luminescent AuNCs embedded in nanogel of chitosan derivative, glycol-chitosan (GC). The as-prepared NCs are highly stable across full pH range and wide salinity range, and have been further used for intracellular sulfide imaging. As illustrated in Scheme 1, the glutathione (GSH) was chosen as thiolate ligand to form Au-thiolate complexes, which were aggregated along with the self-assemble of GC, and finally the AuNCs@GC with strong luminescence were obtained. Due to the confinement of GC nanogel, the emission of the AuNCs@GC was greatly enhanced compared to that of

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bare AuNCs without GC coating. Moreover, given that the GC has a large number of positively charged amino groups; it would play an important role in enrichment of sulfur ion and effectively improve the sensitivity, which demonstrated a limit of detection (LOD) of 2.6 µM in the range of 5.0 - 700 µM for H2S, a remarkable 28-fold improvement on the sensitivity as compared to that of detection method using AuNCs without GC coating as probes. Meanwhile, the as-prepared AuNCs@GC nanoprobes displayed high selectivity toward hydrogen sulfide, which allows for fluorescence imaging and sensing of H2S in living cells with satisfied performance.

Scheme 1. Schematic illustration for the fabrication of AuNCs@GC (A) and fluorescence imaging for hydrogen sulfide in living cells (B).

EXPERIMENTAL SECTION Chemicals and Reagents. Glycol Chitosan (Wako Pure Chemical Industry, Ltd.) was purchased from Invitrogen. NaOH, NaCl, KBr, KI, Na2SO4, KHSO4, NaNO2, NaNO3, Na2S·9H2O, NaHCO3, Na2CO3, NaOAc, Na2HPO4·12H2O, HOAc, NaH2PO4·2H2O, glutathione (GSH) and L-cysteine were purchased from Beijing Chemical Factory (China). HAuCl4·4H2O was supplied by Sinopharm Chemical Reagent Co., Ltd. Fluorescein isothiocyanate (FITC) was purchased from Aladdin. For cell culture, Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffer solution (PBS) were obtained from M&C Gene Technology. Fetal calf serum (FBS) was purchased from Hangzhou Sijiqing Bioengineer Materials Ltd. Methyl thiazolyl tetrazolium (MTT) were purchased from Sigma-Aldrich. All of the above chemicals were used as obtained without further purification. Ultrapure Milli-Q water was used throughout all experiments. Instrumentation. Transmission electron microscopy (TEM) images were obtained by a JEOL JEM-1200EX (200 kV). Dynamic light scattering (DLS) and potential analysis were conducted on a Zetasizer Nano-ZS90 zeta and size analyzer from Malvern. The fluorescence measurements were carried out on a model F-4600 spectrophotometer (Hitachi, Japan). The X-ray photoelectron spectrum (XPS) tests were performed on ESCALAB 250 (Thermo-Fisher Scientific, USA). Cell viability was measured by a Tecan Infinite F50

(Switzerland) plate reader. The further cell imaging results were recorded with an EVOS microscopes system (Life Technologies) with excitation wavelength range at 400-435 nm. Preparation of AuNCs. The AuNCs without glycolchitosan were prepared according to a reported method.37 Briefly, GSH solution (50 mM, 2.40 mL) was added to HAuCl4·4H2O solution (20 mM, 4.0 mL) under gentle stirring at 25 °C. The solution was then heated to 70 °C for 24 h and cooled to room temperature. The AuNCs were obtained and purified by dialysis process and adjusted the solution to 30 mL for further use. Preparation of AuNCs@GC. Firstly, 25 mg of GC was dissolved in 5.0 mL of deionized (DI) water to get the GC stock solution with a final concentration of 5 mg/mL. By diluting this stock solution, different concentrations of GC could be obtained. In a typical case, GSH solution (50 mM, 0.3 mL) was added to GC solution (8 mL), followed by addition of HAuCl4 solution (50 mM, 0.2 mL). Then the pH of the solution was adjusted to 6.0 by NaOH solution (1.0 M). The solution was heated to 80 °C for 6 h and then cooled to room temperature. The AuNCs@GC colloid solution was obtained after centrifugation and then redispersed into 4.0 mL of DI water for later use. Other control experiments were carried out similarly. General fluorescence measurements for in vitro detection of Na2S. A stock solution of Na2S (30 mM) was firstly prepared and diluted to desired concentrations ranging from 5 µM to 10 mM. Then, 100.0 µL of the AuNCs@GC solution (1.5 mg/mL) was mixed with various concentrations of Na2S, and then the resulting solution was diluted to 1.0 mL with Na2HPO4-NaH2PO4 buffer solution (pH = 7.4, 0.02 M). Fluorescence spectra were recorded with excitation wavelength at 365 nm. Cell culture and fluorescence microscopy imaging. HeLa cells were seeded on a 6-well plate, allowed to adhere for 24 h at 37 °C under a 5% CO2 and 95% relative humidity atmosphere. AuNCs@GC with a final concentration of 100 µg/mL was added into each well to incubate for 4 h, and then washed with PBS to remove excess AuNCs@GC. Then the cells were incubated with different concentrations of Na2S (0, 25, 50, 100 and 100 µM), respectively. After incubation for 30 min at 37 °C, the cells were washed with PBS buffer and then imaged by an EVOS fluorescence microscopes system (Life Technologies) with excitation wavelength range at 400-435 nm. For endogenous sulfides imaging, cells were incubated with AuNCs@GC (100 µg/mL) for 4 h, and then incubated with cysteine (200 µM) for another 30 min. Then the media was replaced by PBS and cell imaging was performed with an EVOS fluorescence microscopes system.

RESULTS AND DISCUSSION Synthesis and characterization of AuNCs and AuNCs@GC. As mentioned above, AuNCs@GC were prepared through in situ growth of AuNCs in the presence of GC. However, the pure AuNCs were prepared through a mild reduction of HAuCl4 solution by L-glutathione (GSH) in the absence of GC. To investigate the encapsulation effect of GC on the synthesis of nanoprobes, different concentrations of GC solution were used. According to the transmission electron microscopy (TEM) images in Figure 1A, the AuNCs were well dispersed in water. For the AuNCs@GC, the amount of GC has an impact on the photoluminescence (PL) intensity of


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Analytical Chemistry the resulting nanoclusters, as depicted in Figure S1. With the increase of GC amount, the PL intensity initially increased, followed by decrease and exhibited the highest value with 1.0 mg/mL of GC. It was assumed that a small amount of GC is insufficient to confine AuNCs adequately, displaying a partially crosslink effect for gold clusters (Figure S2B), while excess GC could lead to severely aggregation of polymer itself (Figure S2D), which is also unfavorable for the self-assemble of AuNCs and GC. When a proper amount of GC was utilized, a well-organized self-assembled structure (Figure 1B, Figure S2C) could be obtained.

intensity was almost the same for reaction time of 5 or 6 h, here we chose 5 h as the reaction time. Meanwhile, other properties of AuNCs and AuNCs@GC have also been characterized. According to the dynamic light scattering (DLS) results (Figure 1C), the average size of AuNCs was about 1.5 ± 0.8 nm, after being impregnated into the GC matrix, the average size of AuNCs@GC was increased to about 102.3 ± 7.9 nm. The localization of AuNCs into GC nanogel greatly enhanced the PL intensity and largely prompted the QYs (36.42%), nearly 6-fold of that (6.38%) of AuNCs without GC coating (Table S1). As compared with previously reported hydrophilic AuNCs,26, 31, 33, 38 the as-prepared AuNC@GC displayed satisfied QYs (Table S2). In addition, to better understanding the charge transfer of AuNCs before and after being embedded in the nanogel, X-ray photoelectron spectrum (XPS) experiments were also conducted. As indicated in Figure 1E and F, for the AuNCs, the ratio of Au(0)/Au(I) is 1.03, which means the emissive component of Au(I) species35 is contributed to 49.26% of all the Au atoms in Au clusters. While for AuNCs@GC, the percentage of Au(I) changed into 78.45%, which means the amount of luminescent Au(I)thiolate complex greatly increased. Considering that the introduction of GC is the main reason for the change of the ratio Au(0)/Au(I) of the NCs, we further investigated the amount of GC on the percentage of Au(I)-thiolate complex. As shown in Figure S4, along with the increase of GC from 0.5 to 1.5 mg/mL, the fraction of Au(I) is 67.37%, 78.45% and 78.11%, respectively. The increased percentage of Au(I) might be attributed to the electrostatic interaction and coordination effect of AuNCs and GC, and further resulted in the promoted QY of the AuNCs@GC. 1.0 mg/mL of GC is enough to full coordinate the nanocluster, further increasing the GC has no obvious influence on the fraction of Au(I). As a whole, it can be concluded that the synergistic effect of the nanogel confinement and the increase of Au(I)-thiolate may account for the enhanced emission.

Figure 1. TEM images (A: bare AuNCs, B: AuNCs@GC, GC = 1.0 mg/mL), size distribution (C), photoluminescence spectra and corresponding photographs (D), and XPS (E, F,) analysis of AuNCs and AuNCs@GC, respectively.

In addition, influence of pH conditions and reaction time on the PL intensity of AuNCs@GC was also investigated. In order to get the best self-assembled structure, the proper pH value within the gelation pH range, namely, between the pKa of GC and GSH, was chosen. It appeared that under pH = 6.0, the formed AuNCs@GC had the highest PL intensity while the PL intensity became rather weak when the pH reached 7.0 (Figure S3 A and B). Considering that GSH is a tripeptide with two carboxylic groups (pKa1 = 2.12, pKa2 = 3.53), we inferred the pH value would have an impact on the coordination and reduction capacity of GSH, further influence the fluorescence of the AuNCs and AuNCs@GC. During the experiment we also found white precipitation from the mixture solution of GSH and HAuCl4 when adjusting pH to 7.0, which would impaired the formation of AuNCs, accordingly with diminished the fluorescence. In another aspect, according to Figure S3C and S3D, with the extension of the reaction time, the fluorescence intensity increased continuously. Since the

Figure 2. PL intensity of AuNCs@GC (A and C) and AuNCs (B and D) under different pH (A and B) and salinity conditions (C and D). Data were normalized to controls and statistical analyses were performed with a t-test method. Error bars represent mean ±SD.



For biological applications, the fluorescence stability of the probe is of critical importance. Given the extraordinary water solubility of glycol-chitosan over the full pH range of 0-14 (Figure S5), we infer the AuNCs@GC may have ideal stability than conventional AuNCs. As shown in Figure 2, we investigated the fluorescence evolution under a wide pH and salinity range. In accordance with our hypothesis, the PL intensity of the AuNCs@GC remained stable when the pH adjusted from 0 to 14 (Figure S6, Figure 2A). Meanwhile, there was negligible influence on the fluorescence intensity of AuNCs@GC even the salinity was up to 100 mM (Figure 2C). However, it turned out that the PL intensity of AuNCs without GC was not so steady compared with the AuNCs@GC. Neither in a wide pH condition (Figure 2B) nor salinity range (Figure 2D), the photoluminescence intensity showed an obvious fluctuation trend to some extent. In addition, the photostability as well as stability in biological media of as-prepared AuNCs@GC were further evaluated. As indicated in Figure S7, negligible photoluminescence intensity changes of AuNCs@GC was observed after continuous irradiation under UV light for 600 min, whereas the common organic dye FITC displayed an obvious decrease of photoluminescence within 150 min. Moreover, when the as-prepared AuNCs@GC was incubated with cell culture media such as DMEM and FBS, both the particle size as well as PL intensity were well maintained, again indicating the excellent stability of the AuNCs@GC (Figure S8). We assumed that the observed excellent stability of the asprepared AuNCs@GC was attributed to the good solubility of GC as well as the effective interaction between GC and AuNCs. As control, the Au NCs nanogel prepared merely with chitosan33 displayed weak luminescence under alkaline conditions, since chitosan itself is insoluble at alkaline condition (Figure S5). When the chitosan precipitate out, the confinement enhancement effect for the AuNCs disappear, thus the luminescence would correspondingly decrease. For AuNCs@GC, the luminescence is stable under full pH range. Under acidic conditions, most of the amine groups of GC are protonated, there would be a strong electrostatic interaction between GC polymer and ligands of Au(I)-SH complexes, accounting for the main force for the formation of the AuNCs@GC structure. As the pH increases to alkaline conditions, amine groups on the GC chain would coordinate with metal center through Lewis base- acid interaction, since it has been reported that chitosan and its derivatives can be used as capping ligand to effectively stabilize metal particles.39 Thus, the utilization of GC can effectively keep the nanogel structure and maintain the fluorescence intensity of AuNCs@GC at wide pH range.40

Sulfides sensing. Considering that the GC holds lots of amine groups and the luminescence of AuNCs is closely related to the Au(I)-complex, we assume such nanogel would be suitable for recognition of hydrogen sulfide. To confirm that GC indeed has the effect of enriching sulfurs and enhancing detection sensitivity, we first investigated the potential changes of AuNCs before and after interacting with GC. As shown in Figure 3A, the AuNCs themself are negatively charged and the zeta potential is about -20.2 mV at pH 7.4. Whereas the AuNCs@GC are positively charged and the zeta potential is 46.3 mV, suggesting the nanocomplex has been successfully formed via electrostatic interactions. After adding Na2S to the solution, it clearly depicted that with the increase of sulfur ions, the zeta potential had an obvious change from positive to negative (Figure 3B).

On the other hand, rapidly and completely recognition is rather important for a sensing system, here we investigated the influence of incubation time for the detection system. Seen from Figure S9, the PL intensity of both AuNCs and AuNCs@GC was quenched dramatically soon after addition of Na2S and almost remained unchanged within further 3 h, indicating this detection method is rapid and reliable. Under optimal conditions, we studied the evolution of PL spectra of the AuNCs@GC with Na2S in the range of 5.0-700 µM. Meanwhile, we also tested the response condition of AuNCs as comparison. According to Figure 3C and 3E, there was a stepwise fluorescence quenching of both AuNCs and AuNCs@GC and the fluorescence intensity was quenched linearly along with the increasing concentration of Na2S. For AuNCs (Figure 3D), the low concentration linear regression equation is I1 = 33.44 - 12.86C1 and the high concentration linear regression equation is I2 = 21.50 - 1.12C2, with a correlation coefficient of 0.9974 and 0.9982, respectively. While for AuNCs@GC (Figure 3F), the low concentration linear regression equation is I1 = 65.36 - 0.15C1 and high concentration linear regression equation is I2 = 54.74 - 0.04C2, with a correlation coefficient of 0.9986 and 0.9843, respectively. The limit of detection (LOD = 3σ/K) for Na2S was 72.5 µM for AuNCs in the linear range of 100 µM - 10 mM. As for AuNCs@GC, the LOD value was 2.6 µM in the range of 5.0 700 µM. Impressively, a 28-fold improvement on the sensitivity was achieved after GC encapsulation.

Figure 3. (A) Zeta potential of AuNCs and AuNCs@GC at pH 7.4. (B) Zeta potential changes of the AuNCs@GC (0.15 mg/mL) along with the addition of sodium sulfide. Fluorescence spectra of AuNCs (C) and AuNCs@GC (E) treated with various concentrations of Na2S; Calibration plot of the PL intensity versus the concentration of Na2S for AuNCs (D) and AuNCs@GC (F), respectively.


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Analytical Chemistry

Figure 4. Selectivity tests with coexisting substances in NaH2PO4−Na2HPO4 buffer solution (pH = 7.4, 10.0 mM); Interference concentration: 500 µM; Na2S: 100 µM.

Furthermore, the sulfide sensors frequently suffer from poor selectivity, particularly for thiol-containing species. Here in order to test the selectivity of this developed system for sulfides, a number of anionic species, especially thiol containing compounds were selected as potential interfering substances. Under identical conditions, only sulfide ion gave rise to a significant luminescence quenching of AuNCs@GC, while other species produced negligible fluorescence changes even at the concentration of 5-fold higher than that of sulfide (Figure 4). This may be attributed to the encapsulation effect of GC matrix, which favors for sulfide entering in the nanogel matrix, thus realizes a high selectivity of AuNCs@GC for aqueous sulfides. Due to the GC matrix shielding, large molecules cannot get close to the emission center (AuNCs), and thus hardly caused influence on the luminescence. In another aspect, even though other coexisting small anions can get into the GC matrix, they cannot cause the agglomeration of AuNCs@GC due to the excellent salt tolerance, and thus the luminescence is not influenced by these coexisting salts. The S2− can not only get close to the AuNCs, but also react with the AuNCs and influence the ratio of Au(I)/Au(0), and then quenching the luminescence, which accounts for the good selectivity. Besides, the fluorescence quenching rate of sulfides for AuNCs and AuNCs@GC could also be evaluated by the Stern-Volmer constant (Ks-v) via the Stern-Volmer equation: I0/I = 1 + KS-V Csulflides Here I0 and I denote the PL intensities of the sensing system in the absence and presence of sulfides, respectively. KS-V is the Stern-Volmer constant, which indicates the quenching efficiency between the nanoprobes and sulfides, and Csulflides is the concentration of aqueous sulfides. The calculated values of the quenching constants (KS-V) were found to be 5.9 × 103 and 3.1 × 104 M−1 for AuNCs and AuNCs@GC, respectively, which manifests that the AuNCs@GC is much sensitive to sulfides.

Figure 5. Fluorescence images of Hela cells cultured with Au NCs@GC and different concentrations of Na2S. (A) 0 µM, (B) 25 µM, (C) 50 µM, (D) 100 µM, (E) 200 µM. Scale bar is 20 µm. (F) The relationship between average fluorescence intensity and the added amount of Na2S in A-E correspondingly.

Fluorescence imaging of sulfides in cells. Encouraged by the good sensitivity and selectivity of AuNCs@GC for Na2S sensing, we further applied these nanoprobes for visualizing sulfides in living cells by using HeLa cell lines as a model. Before imaging studies, MTT (methyl thiazolytetrazolium) assay was used to measure the cytotoxicity of these NCs. According to Figure S10, for the AuNCs@GC, the cells exhibited an excellent viability of over 90% when incubating for over 48 h, indicating the ideal biocompatibility and weak cytotoxicity of these nanoprobes. As for cell imaging, HeLa cells were first incubated with AuNCs@GC for 4 h and then treated with buffer containing different amount of Na2S. After incubation for another 30 min, the cells were washed with PBS buffer and imaged by a fluorescence microscope. The fluorescence images of the group treated without Na2S were also taken as controls (Figure 5A). As shown in Figure 5B-E, the fluorescence intensities became weaker and weaker as the concentration of Na2S increased from 25 to 200 µM. Note that the reason for choosing these concentrations is that it was in the range which could elicit physiological responses (10-600 µM).41-43 The cell regions in the visual field were selected as the regions of interest (ROI) and the average fluorescence intensity was further determined and compared via confocal fluorescence microscope (Figure 5F). To further evaluate the feasibility for recognition H2S, 200 µM of L-cysteine was applied to produce endogenously H2S.44, 45 As indicated in Figure S11, clearly, in the presence of L-cysteine, obvious fluorescence changes could be observed. All the results suggest that these nanoprobes can be used for the sensing of S2− in cells.

CONCLUSION In summary, biocompatible AuNCs@GC nanoprobes with enhanced luminescence and excellent stability over a wide range of pH (0 - 14) and salinity have been achieved through in-situ encapsulation of AuNCs in the matrix of GC nanogel. The electro-positivity of glycol-chitosan not only imposed significant influences on the configuration and luminescence enhancement of AuNCs, but also effectively improved the sensing sensitivity towards sulfides. Moreover, these AuNCs@GC nanoprobes showed high selectivity toward Na2S over thiols and other anions. All the merits enabled the AuNCs@GC to be applicable for the sensitive and selective



imaging of sulfides in cells. It is expected that this novel nanocluster fabrication strategy could be potentially employed for construction of other nanoprobes with high stability, good luminescence, and enhanced sensing selectivity.

ASSOCIATED CONTENT Supporting Information Correlation between the PL intensity of AuNCs@GC and GC concentration (Figure S1), TEM images of AuNCs@GC with different amount of GC (Figure S2), effect of reaction pH conditions or reaction time (Figure S3) on PL intensity of AuNCs@GC, Quantum yields (QYs) of AuNCs and AuNCs@GC (Table S1), Comparison of the QYs of AuNCs@GC with previously reported AuNCs (Table S2), XPS analysis of AuNCs@GC (Figure S4), pH effect on the solubility of chitosan and GC (Figure S5), PL intensities and corresponding optical photos of AuNCs@GC under full pH range (Figure S6), Photostability comparison of aqueous solutions of a common dye FITC and AuNCs@GC under continuous UV irradiation (Figure S7), Stability study of AuNCs@GC dispersed in DMEM and FBS (Figure S8), PL stability of AuNCs and AuNCs@GC along with time upon adding sodium sulphide (Figure S9),Cell viability tests of AuNCs@GC (Figure S10), Fluorescence imaging of AuNCs@GC in the presence and absence of cysteine (Figure S11).

AUTHOR INFORMATION Corresponding Author * Leyu Wang [email protected] Telephone: (86)010-64427869 † These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported in part by the National Natural Science Foundation of China (21725501, 21475007, 21675009 and 21505003), the Fundamental Research Funds for the Central Universities (buctrc201706 and buctrc201720). We are also thankful for the support from the Public Hatching Platform for Recruited Talents of Beijing University of Chemical Technology.

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