Iron-Imprinted Single-Atomic Site Catalyst-Based Nanoprobe for Detection of Hydrogen Peroxide in Living Cells

A facile ion-imprinting method (IIM) is used to synthesize the isolated Fe-N-C single-atomic site catalyst (IIM-Fe-SASC), which mimics the natural enzyme-like active site and shows excellent peroxidase-like activity. The ion-imprinting process can precisely control ion at the atomic level and form numerous well-defined single-atomic Fe-N-C sites. The IIM-Fe-SASC has been successfully used as the nanoprobe for in situ H2O2 detection generated from MDA-MB-231 cells. Supplementary Information The online version contains supplementary material available at 10.1007/s40820-021-00661-z.


S1.2 Catalyst Characterization
The materials were characterized by transmission electron microscopy (TEM, Tecnai F20, 200 kV; Philips CM200 UT, 200 kV; JEOL ARM300F, 300 kV), Xray photoelectron spectroscopy (XPS, Escalab 250, Al Kα), and X-ray diffraction (XRD, Rigaku Miniflex 600, 40 kV). X-ray absorption spectroscopy measurement at Fe K-edge was performed by Advanced Photon Source (APS) on the bendingmagnet beamline 12-BM. The radiation was monochromatized by a Si (111) double-crystal monochromator. All spectra were collected in fluorescence mode by a germanium detector. XAS data reduction and analysis were processed by Athena software. The Fe content of the catalyst was evaluated with an Agilent inductively coupled plasma mass spectrometer (ICP-MS). Preparing a 5mg/ml sample aqua regia solution, the sample was gradually dissolved after 3days. Subsequently, the above solution was diluted with nitric acid (3% v/v) and then subjected to the ICP-MS measurement.

S2.1 Peroxidase Activity and Kinetics Assay of Nanoprobe
The 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2'-azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS) and o-phenylenediamine (OPD) were used as substrates for the colorimetric reaction of prepared samples. Moreover, TMB was further used as a typical substrate to study the peroxidase activity of the obtained single-atomic site catalysts (SASCs). Specifically, different amounts of IIM-Fe-SASC, NIM-Fe-SASC, and NIM were dissolved in 0.2 M pH 3.6 NaAc-HAc buffer, respectively. TMB was dissolved in DMSO to 10 mg mL -1 then added 100 L to the above solutions. The obtained mixtures were incubated in dark at 37 o C for 5 min.
Then H2O2 solution was added to the above mixture to a final concentration of 1M, in which one sample was served as background measurement without adding H2O2. The reaction-time curve of IIM-Fe-SASC was plotted using the absorbance at 652 nm against the reaction time. The catalytic activity units (U) were evaluated by detecting the absorbance at 652 nm of a 10 s interval within 700 s. The catalytic activity expressed in units (U) was calculated through the following equation after eliminating background: In which SA is the specific activity (U mg -1 ); V is the volume of the reaction solution (μL); ε is the molar absorption coefficient of TMB substrate (39,000 M -1 cm -1 at 652 nm); l is the optical path length through reaction solution (cm); ΔA/Δt is the initial rate (within 1 min) of the absorbance change (min -1 ) and m is the amount (mg) of SASCs in each assay.
The steady-state kinetic measurements of three peroxidase-like single-atom nanoprobes were measured according to the following steps. Firstly, 50 μL of 1 μg mL -1 samples in NaAc-HAc buffer (pH 3.6) solution was added to each well, various volumes of TMB solution (10 mg mL -1 in DMSO), then certain volumes of H2O2 solution were added to the reaction mixture to a concertation of 1M. The absorbance at 652 nm was immediately recorded at a 10 s interval within 60 s, the initial rates of the chromogenic reaction to different TMB concentrations were obtained and were fitted with Michaelis-Menten model. Furthermore, Michaelis constant Km and Kcat were calculated according the following Michaelis-Menten equation:

where v is the initial rate of the chromogenic reaction, [S] is the TMB concentration and [E] is the concentration of catalysts (M).
To evaluate the temperature stability of the IIM-Fe-SASC, the samples were incubated at different temperatures for 2h, then conducted the experiments at room temperature. For determining pH stability of the IIM-Fe-SASC, we incubated samples at a higher concentration by HAc-NaAc buffers with different pH for 2h, then diluted the samples with pH 3.6 buffer, and then the peroxidase-like activity was tested under standard conditions.

S2.2 Cell Culture and Cytotoxicity Assay
MDA-MB-231 cells were cultured with Dulbecco's modified Eagle medium under oxidative stress conditions supplemented with fetal bovine serum (10%), and then kept in a humid environment with 5% CO2 at a temperature of 37 o C. In vitro cytotoxicity of the material to MDA-MB-231 cells by standard 3-(4,5dimethylthiazo-2-yl)-2,5-di-phenyltetrazoliumromide (MTT) analysis. The MDA-MB-231 cells were incubated in a 96-well plate at a cell density of 1.0×105/mL for 24 hours. The medium was then discarded and treated with Dulbecco's modified Eagle medium for 8 hours, which contained different concentrations of IIM-Fe-SASC nanoprobe or TMB. After that, the cells were incubated with 20 µL MTT (5 mg mL -1 in PBS) for 4 hrs. After the incubation process, 150 µL of DMSO was added to each well and then used a microplate reader to record the optical density at a wavelength of 490nm. Figures   Fig. S1 TEM images of Fe-doped (left) and without doped (right) mesoporous SiO2 precursor