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Biomimetic mineralization guided one-pot preparation of gold clusters anchored 2D MnO2 nanosheets for fluorometric/magnetic bimodal sensing Jianping Sheng, Xingxing Jiang, Liqiang Wang, Minghui Yang, and You-Nian Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05267 • Publication Date (Web): 24 Jan 2018 Downloaded from http://pubs.acs.org on January 24, 2018

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Analytical Chemistry

Biomimetic mineralization guided one-pot preparation of gold clusters anchored 2D MnO2 nanosheets for fluorometric/magnetic bimodal sensing Jianping Sheng,†, ‡ Xingxing Jiang,† Liqiang Wang,† Minghui Yang,*, † You-Nian Liu*, †, ‡,§ †

College of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan 410083, P. R. China Hunan Provincial Key Laboratory of Efficient and Clean Utilization of Manganese Resources, Central South University, Changsha, Hunan 410083, P. R. China § State Key Laboratory for Powder Metallurgy, Central South University, Changsha, Hunan 410083, P. R. China ‡

Supporting Information Placeholder ABSTRACT: A novel fluorometric/magnetic bimodal sensor was reported based on gold nanoclusters (Au NCs) anchored 2D MnO2 nanosheets (Au NCs-MnO2) that synthesized through one-pot biomimetic mineralization process. Bovine serum albumin (BSA) was used as template to guide the formation and assembly of Au NCs-MnO2 under physiological conditions and without use of any strong oxidizing agent, toxic surfactant as well as organic solvent. The fluorescence of Au NCs was first quenched by MnO2 nanosheets. While upon H2O2 introduction, the MnO2 nanosheets can be sensitively and selectively reduced to Mn2+ with enhanced magnetic resonance (MR) signal and rapid recovery of Au NCs fluorescence simultaneously. This dual-modal strategy can overcome the weakness of single fluorescence detection mode. A linear range of 0.06–2 µM towards H2O2 was obtained for the fluorescence mode, whereas the MR mode also allowed detection of H2O2 at a concentration ranged from 0.01 to 0.2 mM. Benefiting from the BSA molecule residual on the product surface, the as-prepared Au NCs-MnO2 displays low cytotoxicity and good biocompatibility. Importantly, the successful application of Au NCs-MnO2 for analysis of H2O2 in biological samples and cells indicates that the integration of Au NCs fluorescence with Mn2+ MR response provides a promising bimodal sensing platform for H2O2 in vivo monitoring.

Fluorescence nanoprobes have received wide attention in the detection of important physical molecules and ions, owing to the superiorities of fast response, high throughput and sensitive analysis.1,2 MnO2, especially 2D MnO2 nanosheet-based fluorescence nanoprobes have attracted particular interest due to their large specific surface area and superior light absorption capability.3 Typically, fluorescent molecules were anchored onto MnO2 nanosheets to construct smart platforms for sensing applications.4 Structural combination of MnO2 nanosheets with other materials affords newly formed composites enhanced or synergistic properties, which can largely extend their practical application.4-6 For example, Yuan et al. reported a MnO2 nanosheets modified upconversion nanoparticles for rapid and sensitive detection of glucose in human serum and whole blood.1 A nanocompsite of MnO2 nanosheets anchored with upconversion nanoprobes was synthesized by Fan et al., which showed high stimuli-responsive imaging/therapy for solid tumors.2 Zhao et al. also synthesized a MnO2 nanosheet-aptamer nanoprobe for target-cell-activated tumor cell imaging.7 However, most of these existed hybrid materials are fabricated via post assemble process, which is hybridizing the as-prepared fluorescent materials and MnO2 nanosheets via the unspecific interaction, resulting in poor stability of the composites. Besides, it is inevitable to use strong oxidizing reagent (e.g. KMnO4), nocuous surfactants and/or organic solvent and etc. in the conventional synthesis of MnO2 nanosheets, which is not beneficial to bioassays. Furthermore, the previously reported MnO2 based sensors are

almost used for single-mode detection, while single optical sensing displays poor spatial resolution and cannot provide comprehensive information for accurate diagnosis and treatment. Therefore, the combination of fluorescence sensing with other high penetrable and spatial resolution imaging techniques, such as magnetic resonance imaging (MRI), is significant not only for molecular imaging, but also for clinical diagnosis with high precision and accuracy.8 As we all know, MnO2 can be easily decomposed into Mn2+, which has strong MRI signal.7,9,10 Thus, a novel fluorescence/magnetic resonance bimodal nanoprobe can be constructed based on MnO2 nanosheets. This procedure could be an effective way to improve detection accuracy and expand practical applications, since MRI is useful for surgical planning, meanwhile, fluorescence imaging is helpful to guiding the operating surgeon during the procedure. However, to improve the tedious synthetic procedure of this bimodal nanoprobe still remains great challenging. To date, protein based biomimetic mineralization has attracted much attention for synthesis of various nanostructures under facile reaction conditions.11,12 Our previous work has demonstrated that using proteins can control the synthesis of biocompatible metal sulfide nanoparticles by simply mixing metal ion and proteins under alkaline conditions.13 Liu et al. also reported that MnO2 nanoparticles can be obtained through incubating Mn2+ with bovine serum albumin (BSA) aqueous

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solution under alkaline conditions.10,14 However, there are few reports on the protein guided synthesis of 2D MnO2. In this work, 2D MnO2 nanosheets were obtained via BSA based biomimetic mineralization for the first time. This proposed strategy can avoid the usage of strong oxidizing reagent of KMnO4, surfactants and organic solvents. Notably, BSA has also been proved capable of templating fluorescent metal nanoclusters (MNCs), such as gold nanocluster (Au NCs), silver nanocluster (Ag NCs) and copper nanocluster (Cu NCs).15-17 However, it is still of great challenge to realize the one-step preparation of MNCs anchored-MnO2 nanocomposite platform without inter-particle “contaminations”. This is particular the case for MNCs, their emission tend to be quenched by metal ions.18,19 Enlighten by the above breakthrough, we believe that BSA may work as a template to guide the formation of MNCs-2D MnO2 nanocomposites through a onestep synthesis process under accessible conditions. More significantly, the BSA molecule residual can improve the biocompatibility of the MNCs-2D MnO2 nanocomposites. Hydrogen peroxide (H2O2) is one of the most significant chemicals that widely used in clinical and pharmaceutical areas.20 Particularly, as an important reactive oxygen species,21 H2O2 is the intermediate product of various physiological process that highly related to many diseases such as myocardial infarction, Alzheimer’s disease, atherosclerosis, cancer and etc.22-24 Therefore, diverse methods such as chemiluminescence, spectrophotometry, titrimetry and electrochemical methods have been employed to detect H2O2.24-28 However, these methods still cannot satisfy the practical requirements for H2O2 sensing in different diversions, especially for in vivo sensing. Therefore, unconventional strategies are desired for rapid and sensitive detection of H2O2. Considering 2D MnO2 nanosheets can be disrupted by H2O2 into Mn2+, giving an enhanced MRI signal, we report here fluorometric/magnetic bimodal sensor for H2O2 based on Au NCs anchored 2D MnO2 nanosheet (Au NCs-MnO2). To the best of our knowledge, there are few reports about MRI sensing of H2O2 based on Mn2+. Our strategy aims at synthesizing Au NCs-MnO2 nanosheets by one-pot and “green” biomimetic mineralization guided method utilizing BSA as a pH and temperature responsive template. The obtained Au NCs-MnO2 nanocomposites demonstrate enhanced performances for detection of H2O2. In this sensing platform, the fluorescence of Au NCs can be effectively quenched by 2D MnO2. In the presence of H2O2, MnO2 can be disrupted into Mn2+, which restores the fluorescence of the Au NCs and results in the increase of MRI signal. The Au NCs-MnO2 was also applied for monitoring change of H2O2 in living cells and real serum samples. In addition, this convenient procedure will also be suitable for preparation of other fluorometric metal nanoclusters anchored 2D MnO2 nanosheets in wide prospect. EXPERIMENTAL SECTION Reagents and materials. All chemicals were of analytical grade and used without further purification. Milli-Q water was used through the whole experiments. BSA, NaOH, H2O2, HAuCl4 and Mn(NO3)2 were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Apparatus. The size and morphology of the nanocomposites were characterized by transmission electron microscopy (TEM; FEI Titan G2 60-300 with spherical aberration correction, FEI, USA). Powder X-ray diffraction spectra were collected on a Rigaku D/max-2500 X-ray diffractometer (Rigaku, Japan) at room temperature. Atomic force microscope (AFM;

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NanoManVS, Veeco, USA) was used to evaluate the morphology and height of the nanocomposites with the tapping mode using commercial silicon probes. It was operated at ambient temperature (25±1 oC) with a relative humidity of 20%. The samples were directly deposited on a freshly cleaved mica slide (10×10 mm) and each AFM analysis was performed over a 1 µm×1µm area. The images were recorded at a scan rate of 2 Hz. The captured raw images were analyzed by the Nanoscope scan analysis software (Bruker) and flattened to remove any underlying surface curvature. X-ray photoelectron spectroscopy (XPS, Thermo Fisher, USA) was carried out using a Thermo Fisher ESCALAB 250Xi with Al Kα excitation (150 W, Mono 500 µm), and the energy step size and pass energy were 0.05 eV and 30 eV, respectively. Binding energy calibration was based on C 1s at 284.8 eV. The mass fraction of Mn element in the Au NCs-MnO2 nanocomposite was determined by flame atomic absorption spectrophotometry (Hitachi Z2000, Japan). Fluorescence measurements were performed on a Luminescence Spectrometer (LS-55, Perkin Elmer, USA). The absorbance measurements were carried out on a Shimadzu UV-2450 spectrophotometer equipped with a quartz cuvette (Shimadzu, Japan). The intracellular fluorescence imaging was carried out on an inverted fluorescence microscope (IX 83, Olympus, Japan). The measuring of longitudinal relaxation time (T1) was carried out with a 0.5 T on NMI20-Analyst NMR Analyzing & Imaging system (Niumag Corporation, Shanghai, China). Scheme 1. Schematic representation for synthesis of Au NCs-MnO2 nanosheets and H2O2 sensing

Synthesis of Au NCs-MnO2 nanosheets. Au NCs-MnO2 nanosheets were prepared through a one-pot procedure. Typically, 100 µL of BSA solution (250 mg mL–1) was mixed with 450 µL of water. Then, 200 µL of Mn(NO3)2 solution (50 mM) and 200 µL of aqueous HAuCl4 solution (25 mM) were added dropwise into the BSA solution. Thereafter, 50 µL of NaOH solution (5 M) was added into the mixture. The mixture was incubated at 37 oC for 12 h with vigorous stirring. After cooling to room temperature, the mixture was purified by dialysis (molecular weight cut-off MWCO = 14 kDa) against water for 24 h to remove excess salts and NaOH. The final solution was stored at 4 oC. Synthesis of Au NCs. Au NCs were synthesized similar to that of Au NCs-MnO2 nanosheets without addition of Mn(NO3)2 (see Supporting Information for experimental details). Synthesis of MnO2 nanosheets. MnO2 nanosheets were synthesized similar to that of Au NCs-MnO2 nanosheets without addition of HAuCl4. (see Supporting Information for experimental details). Detection of H2O2 in fluorescence mode. Typically, a stock solution of H2O2 (100 µM) was prepared in a phosphate buffered saline solution (PBS, pH 7.0) and stored in 4 oC, from which various concentrations of H2O2 were prepared by


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Analytical Chemistry serial dilution. Aliquots (400 µL) of freshly prepared PBS solution (pH 7.0) containing 625 µM Au NCs-MnO2 (based on the content of Mn) were used for H2O2 detection, into which 100 µL of H2O2 solution in PBS (pH 7.0) with different concentrations were added. The mixture was incubated at room temperature for 30 min. Thereafter, the emission of Au NCs from 500–800 nm was recorded by a luminescence spectrometer when excited at 470 nm. Response of Au NCs-MnO2 nanosheets as MRI contrast agents to H2O2. The T1-weighted signals of different concentrations of Au NCs-MnO2 nanosheets (based on the content of Mn) treated with or without 1 mM of H2O2 were measured at 0.5 T on a NMI20-Analyst NMR Analyzing & Imaging system. In detail, 2 mM stock solution of Au NCs-MnO2 was prepared in PBS (pH 7.0), from which various concentrations of Au NCs-MnO2 were prepared by serial dilutions. Aliquots (400 µL) of PBS solution (pH 7.0) containing various concentrations of Au NCs-MnO2 were mixed with another 100 µL of PBS solution (pH 7.0) containing 1 mM H2O2 or pure PBS solution (pH 7.0). The mixture was incubated at room temperature for 30 min before MR test. The responses of Au NCs-MnO2 to different concentrations of H2O2 were also tested under the similar test conditions. A stock solution of H2O2 (5 mM) was prepared in PBS (pH 7.0), from which various concentrations of H2O2 were prepared by serial dilution. Aliquots (400 µL) of freshly prepared PBS solution (pH 7.0) containing 1.2 mM Au NCs-MnO2 (based on the content of Mn) were mixed with 100 µL of PBS solution (pH 7.0) containing various concentrations of H2O2. The mixture was incubated at room temperature for 30 min before MR test. Specificity investigations. To evaluate the selectivity of the as-prepared Au NCs-MnO2 nanocomposite for H2O2, interferents including common metal ions (K+, Ca2+, Mg2+, Na+, Fe3+), amino acids (tryptophane (Try), histidine (His), phenylalanine (Phe), aspartic acid (Asp), glycine (Gly), glutamic acid (Glu)), and other biological compounds (BSA, glucose (GU), dopamine (DA), uric acid (UA), ascorbic acid (AA)) that usually coexist with H2O2 were studied under fluorescence/MR modes. Cell cytotoxicity study. In vitro cytotoxicity of the Au NCs-MnO2 nanocomposite was evaluated using 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay in 4T1, HeLa and L929 cells. Briefly, 200 µL of cells (5 × 103 mL–1) was seeded into a 96-well plate and allowed to adhere in a 5% CO2 incubator at 37 oC overnight. After that, the culture medium was replaced by 200 µL of new culture medium which contains various concentrations of Au NCs-MnO2 nanocomposites (10, 30, 50, 70, 100, 150 and 200 µg mL–1, the Au NCs-MnO2 concentration was determined by discrepancy of a certain volume of Au NCs-MnO2 before and after freeze-drying). After 24 h culturing, the medium was replaced by 100 µL of MTT (0.5 mg mL–1 in culture medium) and incubated for another 4 h. Then the medium was removed, and 100 µL of dimethyl sulfoxide (DMSO) was added into each well to dissolve formazan crystals. After full dissolution, the absorbance at 490 nm (OD490 nm) and 630 nm (OD630 nm) were measured on a microplate reader (Bio-Tek Elx800, USA). Cell viability was calculated by the following equation: Cell viability =

/ − / × 100% (1) / − /

Intracellular H2O2 detection. 1.0 mL of HeLa cells (5 × 103 mL–1) were seeded into a 24-well plate and allowed to

adhere in a 5% CO2 incubator at 37 oC overnight. After that, the old culture medium was replaced by 1.0 mL of new culture medium that contains 50 µM of H2O2. After 4 h, the culture medium was removed and the cells were washed by PBS for three times. The cells were then incubated with 1.0 mL of new culture medium containing 500 µM of Au NCs-MnO2 nanocomposites for another 4 h. The final cells were washed with PBS (pH 7.0) and imaged using an inverted fluorescence microscope. The MR images of cell lysis solution were also carried out on an NMI20-Analyst NMR Analyzing & Imaging system under 0.5 T. Detection of H2O2 in serum sample. A 5% of fetal bovine serum (FBS) PBS stock solution was prepared by diluting FBS with PBS (pH 7.0). Then the diluted FBS solution was incubated with 500 µM of Au NCs-MnO2 and different concentrations of H2O2 (0.5, 1.0 and 1.5 µM) for 30 min. After that, luminescence spectrometer was used to record the emission of the FBS solution. RESULTS AND DISSCUSSION Preparation and characterization of the Au NCs-MnO2 nanosheets. Proteins, which play a key role in biomimetic mineralization, are with diverse functional groups, like – COOH, –NH2, –SH, and can be driven to form versatile assembles by tuning chemical surroundings (e.g. pH, temperature, protein concentration and incubation time).29 This feature enables them excellent templates to guide the formation of diverse nanostructures.30,31 The schematic representation of the one-pot synthesis of Au NCs-MnO2 and the sensing of H2O2 is illustrated in scheme 1. First, Mn2+ and [AuCl4]– were mixed with BSA aqueous solution under vigorous vortexing. BSA can seize Mn2+ and [AuCl4]– with the help of carboxyl groups and thiol groups to form BSA-Mn and BSA-Au complexes. Then, the nucleation and growth of 2D MnO2 and Au NCs were triggered when NaOH was added into the above solution. BSA acts here as both template and reductant in the synthesis process. On one hand, the Mn ions that sequestered by BSA can initially turn into Mn(OH)2 through hydrolysis reaction, and the intermediate Mn(OH)2 can then be oxidized to MnO2 with the help of dissolved oxygen. The whole process can be described by the following equation32,33: 2Mn2+ + 4OH– + O2 → 2MnO2 + 2H2O (2) On the other hand, the [AuCl4]– ions that entrapped by BSA can be reduced to form Au NCs under the influence of reductive BSA molecules. Further interactions between 2D BSAMnO2 and BSA-Au NCs resulted in Au NCs-MnO2 assemblies with the help of BSA. Here, BSA plays another important role as “glue” in this progress. The obtained Au NCs-MnO2 was characterized by various microscopic and spectroscopic methods. The morphology and structure of the Au NCs-MnO2 were characterized by TEM, high-angle annular dark-field scanning TEM (HAADFSTEM) and atomic force microscopy analysis (AFM). TEM shows the Au NCs-MnO2 nanocomposites are flat 2D nanosheets with dimensions about 50–100 nm (Figure 1a). The mapping and energy-dispersive X-ray spectroscopy (EDX) confirm the presence of Mn, Au, N, O and S elements (Figure 1a and Figure S1b). High-magnification TEM (Figure 1b) and HAADF-STEM (Figure S1a) also show that Au NCs are successfully interspersed on the surface of 2D MnO2 nanosheets with a mean size of 1.9 ± 0.43 nm. The distinct lattice fringe of 0.212 nm with β angle of 103o was observed in high-resolution TEM (HRTEM, Figure 1c), which is consistent with the (-112) plane of the birnessite-type MnO2.


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AFM analyses in Figure 1d reveals the thickness of the Au NCs-MnO2 nanosheets is about 1 nm. The structure and phase purity of the Au-MnO2 samples

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quench the fluorescence of Au NCs. As shown in Figure 2b and c, the fluorescence intensity of Au NCs can indeed be gradually quenched with the increasing amount of 2D MnO2. However, once H2O2 was added, the fluorescence intensity of Au NCs was rapidly recovered because of the reduction of MnO2 by H2O2 (Figure 2d). The fluorescence intensity of Au NCs reached a plateau within 5 min at room temperature. (Figure S2) The reduction of MnO2 to Mn2+ by H2O2 was verified by UV-Vis spectra. The absorbance intensity of MnO2 was gradually reduced along with the increase of H2O2 concentrations. The linearity of absorbance intensity at 400 nm to H2O2 concentration is illustrated in inset of Figure S3a and S3b, indicating the good correlation between the absorbance intensity of MnO2 and the concentrations of H2O2. Whereas, the response of single Au NCs to H2O2 was negligible within 0–20 µM (see Figure S4) which indicating that the Au NCs just acted as a fluorescence reporter and MnO2 nanosheets acted as fluorescence quencher and H2O2 recognizer.

Figure 1. (a) TEM image of Au NCs-MnO2 and corresponding mapping image. (b) High-magnification TEM image of Au NCs-MnO2. (c) HRTEM image of Au NCs-MnO2. (d) Atomic force microscopy image and corresponding line-scan profile of Au NCs-MnO2. (e) XRD pattern of Au NCs-MnO2. (f) XPS survey of Mn 2p spectra for Au NCs-MnO2. (g) XPS survey of Au 4d spectra for Au NCs-MnO2. were examined by X-ray powder diffraction (XRD, Figure 1e). The diffraction peaks at about 12.5, 25.2 and 37 o (2θ) from the Au-MnO2 nanosheets matched the standard XRD pattern of birnessite-type MnO2 crystal (JCPDS 43-1456, a 5.175 Å, b 2.849 Å, c 7.338 Å). No other peaks were observed, indicating high purity of the synthesized MnO2. XPS was applied to investigate the chemical state of manganese element in MnO2 nanosheets (Figure 1f). Two characteristic peaks at 653.9 and 642 eV are observed, which are correspond to the Mn (IV) 2p2/3 and Mn (IV) 2p1/2 spin–orbit peaks of MnO2, respectively. The spin-energy separation of 11.9 eV is also consistent with previous observations, which suggests the Mn4+ ions are dominant in the product. In Figure 1g, we can also observe two Au 4f binding-energy peaks at 87.61 and 83.93 eV, which are consistent with the previous report for Au. These data proved the successful synthesis of Au NCsMnO2 flat 2D nanosheets. The synthesized nanocomposites were employed for H2O2 detection. This novel sensing platform consists of Au NCs and 2D MnO2, where Au NCs serve as a fluorometric reporter and MnO2 works as fluorescence quencher and H2O2 recognizer. The H2O2 detection mainly involves the fluorescent quenching of Au NCs by the 2D MnO2 and the decomposition of 2D MnO2 by H2O2. The disruption of MnO2 to Mn2+ by a small amount of H2O2 is based on the chemical redox reaction which can be represented as the following equation1,14: MnO2 + H2O2 + 2H+ → Mn2+ + 2H2O + O2 (3) As shown in Figure 2a, the UV-visible absorption spectra of the synthesized 2D MnO2 nanosheets (black line) shows a strong broad absorption bands (200–800 nm), originating from the d-d transition of Mn ions in the MnO6 octahedra. The prepared Au NCs also show typical dual emissions at 448 and 660 nm (blue line) when excited at 470 nm. As seen from Figure 2a, the absorption spectrum of 2D MnO2 nanosheets is well overlapped with the emission spectra of Au NCs. Due to the well match of the absorption/emission spectra of 2D MnO2 nanosheets and Au NCs, 2D MnO2 nanosheets can effectively

Figure 2. (a) UV-vis absorption spectrum of 2D MnO2 (black line) and fluorescence emission spectrum of Au NCs (blue line). (b) Normalized fluorescence emission spectra of the Au NCsMnO2 at different MnO2 concentrations (5 mM of Au NCs). (c) Fluorescence quenching efficiency versus concentrations of 2D MnO2. Inset: photographic image of the aqueous Au NCs-MnO2 solutions. (d) Fluorescence spectra of Au NCs-MnO2 solution in the presence (red line) and absence (black line) of H2O2. (e) ∆1/T1 versus Mn concentration for Au NCs-MnO2 solution (black line) and Au NCs-MnO2 treated with H2O2 (red line). (top portion of Figure 2f) T1-weighted MR images obtained from Figure 2e. (bottom portion of Figure 2f) MR images of the solution containing different H2O2 concentrations (0.01, 0.04, 0.1, 0.2, 0.3, 0.6 and 1.0 mM). The Au NCs-MnO2 concentration is 1.2 mM.

Mn atoms in 2D MnO2 are coordinated with six oxygen atoms to construct MnO6 octahedra. The Mn atoms that restricted in MnO6 octahedral geometry are shielded from aqueous environment, which results in no contribution to the longitudinal relaxation of protons. Nevertheless, MnO2 would be de-


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Analytical Chemistry composed into Mn2+ in the presence of H2O2, and the produced Mn2+ with five unpaired 3d electrons is a great T1shortening agent in MR imaging. Thus, free Mn2+ is a high T1weight contrast agent compared with 2D MnO2 nanosheets. To evaluate the effectiveness of Au NCs-MnO2 nanocomposites as a H2O2-responsive MRI contrast agent, longitudinal relaxation rate (1/T1) of Au NCs-MnO2 in the presence and absence of H2O2 were studied. The Au NCs-MnO2 nanosheets treated with H2O2 show much stronger enhancement of T1-weighted MRI signal than the untreated ones (Figure 2e and f). Besides, the T1-weight MR images were gradually lightened with increasing of the H2O2 concentrations, indicating the positive correlation between MRI signal and concentrations of H2O2 (Figure 2f below). Considering the high tissue penetration capability of MRI detection, the as-prepared Au NCs-MnO2 has great potential in biological applications. As control, longitudinal relaxation rate (1/T1) of the single MnO2 nanosheets in the presence and absence of H2O2 were also studied, and a similar result was obtained, which suggests that the enhancement in relaxation is attributed to Mn rather than Au (Figure S5).

Figure 3. (a) Fluorescence emission spectrum of Au NCsMnO2 probe with different concentrations of H2O2 (0.06, 0.1, 0.2, 0.4, 0.6, 1.0 and 2.0 µM). The concentration of MnO2 was fixed at500 µM. Inset: the change trend of F/F0–1 with different H2O2 concentrations and the corresponding photograph of Au NCs-MnO2 solutions. (b) Relationship between F/F0–1 and the different H2O2 concentrations. F and F0 are fluorescence intensities of Au NCs-MnO2 probe at 660 nm in the presence and absence of H2O2, respectively. Detection of H2O2. The responses of the Au NCs-MnO2 nanocomposites to different concentrations of H2O2 were studied. The fluorescence intensity was gradually recovered along with increasing concentrations of H2O2 (Figure 3a). In the inset of Figure 3a, it can be seen that the reddish fluorescence of Au NCs is gradually enhanced with the increasing concentrations of H2O2. To visualize the relationship between fluorescence restoration and the concentrations of H2O2 added, the fluorescence intensity of Au NCs-MnO2 solution at 660 nm in the presence (F) and absence (F0) of H2O2 were calculated (F/F0–1). These results were fitted with the concentration of H2O2 added (see Figure 3b). The fluorescence enhancement is found to be linearly dependent on the concentration of H2O2 ranging from 0.06 – 2 µM with the correlation coefficient of 0.995. Based on signal-to-noise of 3 criteria,6 the limit of detection for H2O2 was calculated to be 53 nM. MRI performance of Au NCs-MnO2. Longitudinal relaxation rate (1/T1) before and after reaction with H2O2 were examined to evaluate the effectiveness of Au NCs-MnO2 as a H2O2 activated MRI contrast agent. The MR response of the Au NCs-MnO2 nanocomposites at different H2O2 concentrations was investigated. As shown in Figure 4a and b, the longitudinal relaxivity curves exhibit a good enhancement in relax-

ation with an increase in H2O2 concentration. The brightness of MRI images was increased obviously (inset of Figure 4a). The MRI signal shows a linear dependence on the H2O2 concentration, and the relationship is linear in the range of 10 – 200 µM. Selectivity performance of Au NCs-MnO2. To assess the specificity of Au NCs-MnO2 towards H2O2, the influence of

Figure 4. (a) 1/T1 versus H2O2 concentration curves of the Au NCs-MnO2 solution treated with different concentrations of H2O2 (the Au NCs-MnO2 concentration is 1.2 mM). Inset: T1weight MR images of the Au NCs-MnO2 in the presence of H2O2 (0.01, 0.04, 0.06, 0.1, 0.2, 0.3, 0.6 and 1.0 mM). (b) Relationship between 1/T1 and the different H2O2 concentrations (0.01, 0.04, 0.06, 0.1 and 0.2 mM). different metal ions (K+, Ca2+, Mg2+, Na+, Fe3+), amino acids (tryptophane (Try), histidine (His), phenylalanine (Phe), aspartic acid (Asp), glycine (Gly), glutamic acid (Glu)) and biomolecules (BSA, glucose (GU), dopamine (DA), uric acid (UA), ascorbic acid (AA)) which usually coexist with H2O2 were examined. The results are presented in Figure 5. The Au NCsMnO2 nanosheets reveal a remarkable increase of fluorescence and relaxivity responses toward H2O2. In contrary, no obvious changes could be observed with other compounds. Therefore, Au NCs-MnO2 can provide a highly selective and sensitive approach for H2O2 detection.

Figure 5. (a) Fluorescence and (b) relaxivity responses of the Au NCs-MnO2 to different interferents (0.3 mM metal ions and biomolecules; 1 mg mL–1 of BSA; 5 µM and 0.3 mM of H2O2 for fluorescence and relaxivity responses respectively).

Biocompatibility and sensing of the intracellular H2O2 levels. BSA molecules, which covered on the surface of Au NCs-MnO2 nanocomposites during the preparation process is beneficial for improving the biocompatibility of the nanocomposite. Cytotoxicity of Au NCs-MnO2 was evaluated by measuring cell viability using MTT assay. As shown in Figure 6, no apparent loss of cell viability (˂ 10%) was observed after 24 h of exposure to the Au NCs-MnO2 nanosheets at concentrations below 100 µg mL−1. The relative viability of 4T1, HeLa and L929 cells can still retain up to 85% even at a concentration of 200 µg mL−1 of Au NCs-MnO2. The results indicate that the Au NCs-MnO2 nanocomposite have low cytotoxicity and good


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biocompatibility, which is beneficial for biological applications. The detection of intracellular H2O2 levels was also carried out in living mammalian cells. Considering the extremely low concentration of H2O2 in HeLa cells owing to non-anoxic culture environment, we introduced extraneous H2O2 into HeLa cells through incubating the HeLa cells with H2O2. The HeLa cells were then incubated with 500 µM of Au NCs-MnO2 for another 4 h. The fluorescence images were obtained on an inverted fluorescence microscope (Figure 6b-e). Obvious red emission can be seen in HeLa cells, whereas there was almost no fluorescence signal for the control group which only incubated with Au NCs-MnO2 without introducing H2O2. The corresponding MR images were also obtained (inset of Figure 6de). The change of brightness can be observed, which attributed to the increase of intracellular H2O2 concentration. The good biocompatibility and feasibility of intracellular detection implies that the Au NCs-MnO2 nanosheets have potential for in vivo monitoring of H2O2.

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metic mineralization guided method utilizing BSA both as template and as reductant. The H2O2 assay is based on a fluorescence/MR dual “turn on” detection process. The Au NCs serve as a fluorescence probe and the MnO2 nanosheets are used as both a quencher and a recognizer in the sensing platform. The MnO2 nanosheets which quenched the fluorescence intensity of Au NCs can be selectively and sensitively reduced to Mn2+ by H2O2, along with the subsequent fluorescence recovery of Au NCs and the enhancement of MR signal due to generation of Mn2+. A linear range of 0.06–2 µM to H2O2 was obtained in the fluorescence mode, whereas the MR mode allowed detection of H2O2 at a concentration range from 10– 200 µM. The dual-modal detection can efficiently overcome the shortcomings of single fluorescence detection mode. The successful detection of H2O2 in cells indicated the sensor can be applied for in vivo testing and find wide applications. Our proposed synthetic strategy will also be suitable for preparation of other fluorometric metal nanoclusters anchored 2D MnO2 nanoprobes in wide application.

ASSOCIATED CONTENT Supporting Information

Figure 6. (a) Cell viability of 4T1, HeLa and L929 cells after incubated with different amount of Au NCs-MnO2 for 24 h. Fluorescence microscopy images of HeLa cells incubated without (b and d) and with (c and e) Au NCs-MnO2. And the MRI images of HeLa cells incubated without (inset of d) and with (inset of e) Au NCs-MnO2. Detection of H2O2 in biological samples. The potential clinical application of the Au NCs-MnO2 was further investigated by analyzing H2O2 in serum samples. Standard addition experiments were carried out, and three concentrations of H2O2 were added into the serum samples. After incubating with Au NCs-MnO2, the fluorescence signal were detected and analyzed. As listed in Table 1, we can see that the recoveries range from 95.3% to 98.2%, and a relative low standard deviation (RSD) is obtained for each sample. These results demonstrated the applicability of this novel Au NCs-MnO2 for H2O2 monitoring in biology samples. Table 1. Determination of the H2O2 levels in serum samples using Au NCs-MnO2 nanocomposites added

found

Recovery

/µM

/µM

/%

/%

1

0.5

0.48

95.3

1.5

2

1.0

0.97

97.4

1.2

3

1.5

1.47

98.2

2.1

sample

RSD

We compared the performance of our fluoresence/magnetic bimodal nanoprobe with the fluorescence probes reported in recent years (Table S1). It indicates our method holds more advantages in facile synthesis, rapid response, low LOD and dual model detection for wider application. CONCLUSIONS A fluorometric/magnetic bimodal strategy for H2O2 detection utilizing Au NCs-MnO2 nanosheets as a sensing platform was constructed. The nanosheets were synthesized by biomi-

The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. HAADF-STEM images and corresponding EDX spectra of Au NCs-MnO2 (Figure S1); UV-vis absorption spectrum of MnO2 nanosheets with different concentration of H2O2 (Figure S2); longitudinal relaxation rate (1/T1) of the single MnO2 nanosheets in the presence and absence of H2O2 (Figure S3). (PDF)

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (M. Yang) *E-mail: [email protected] (Y.-N. Liu)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (No. 21636010, 21476266 and 21575165), the State Key Laboratory of Powder Metallurgy, Central South University, Changsha, China, and the Hunan Provincial Science and Technology Plan Project, China (No. 2016TP1007).

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