Tuning Atomically Dispersed Fe Sites in Metal–Organic Frameworks Boosts Peroxidase-Like Activity for Sensitive Biosensing

Highlights The two functional groups (nitro and amino) were introduced into MIL-101(Fe) for tuning the atomically dispersed metal active sites. Benefiting from both geometric and electronic effects, the nitro-functionalized MIL-101(Fe) shows a superior electronic structure of active sites and low reaction energy barrier for the HO* formation. Nitro-functionalized MIL-101(Fe)-based biosensor was successfully employed to detect acetylcholinesterase activity and organophosphorus pesticide. Electronic supplementary material The online version of this article (10.1007/s40820-020-00520-3) contains supplementary material, which is available to authorized users.


S1 Instruments
Transmission electron microscopy (TEM) experiments were performed using a FEI Talos F200x (super-x). The element contents were obtained by inductively coupled plasma optical emission (ICP-OES) spectrometry (Agilent 8800). Powder X-ray diffraction (XRD) patterns were carried using a Tensor 27. The functional groups were analyzed using a NEXUS870 FT-IR spectrometer. X-ray photoelectron spectroscopy (XPS) measurements were used by a VG Multilab 2000 (Thermo Fisher, USA). Electron paramagnetic resonance (EPR) measurements were obtained by an EMXmicro-6/1 (Bruker, Germany). Ultrapure water was obtained from a Milli-Q purification system (Millipore, MA, USA). All the UV-vis and fluorescence spectra were obtained from a multimode reader (Tecan Spark, Switzerland).

S2.1 Preparation of MIL-101
FeCl3•6H2O (675 mg, 2.5 mmol) and terephthalic acid (206 mg, 1.25 mmol) were dissolved in 15 mL N,N-dimethylformamide (DMF) and then the solution was vigorously stirred for 1 h. After that, the mixture transported to a Teflon-lined autoclave for 15 hours at 110 °C. The orange products were washed with DMF and ethanol three times. Finally, the solvent was dried overnight at 80 °C to obtain the powder.

S2.2 Preparation of NO2-MIL-101
FeCl3•6H2O (675 mg, 2.5 mmol) and 2-nitroterephthalic acid (264 mg, 1.25 mmol) were dissolved in 15 mL DMF and then the solution was vigorously stirred for 1 h. After that, the mixture transported to a Teflon-lined autoclave for 12 h at 110 °C. The orange products were washed with DMF and ethanol three times. Finally, the solvent was dried overnight at 80 °C to obtain the powder.

S2.3 Preparation of NH2-MIL-101
FeCl3•6H2O (675 mg, 2.5 mmol) and 2-aminoterephthalic acid (226 mg, 1.25 mmol) were dissolved in 15 mL DMF and then the solution was vigorously stirred for 1 h. After that, the mixture transported to a Teflon-lined autoclave for 12 h at 110 °C. The orange products were washed with DMF and ethanol three times. Finally, the solvent was dried overnight at 80 °C to obtain the powder.

S2.4 Specific Activity of Nanozymes
The specific activity (SA), which is defined as activity units per milligram of nanozyme, was evaluated in different concentrations of nanozymes [S1]. The nanozyme activity (units) was calculated using Eq. (S1): bnanozyme is the catalytic activity of nanozyme expressed in units. V is the total volume of the reaction solution (μL); ε is the molar absorption coefficient of the colorimetric TMB (39,000 (M -1 cm -1 ). l is the path length of light traveling in the cuvette (cm); A is the absorbance value; and ΔA/Δt is the initial rate of change in absorbance at 652 nm min -1 .
Calculate the SA of the nanozyme (U mg -1 ) by nanozyme: ananozyme = bnanozyme/[m]. Where ananozyme is the SA expressed in units per milligram (U mg -1 ) nanozymes, and [m] is the nanozyme weight (mg) of each assay.

S2.5 Verification of Intermediate (•OH)
The blue methylene blue (MB) could be degraded to the colourless products in the presence of •OH. Therefore, MB was usually employed to verify the existence of •OH by colorimetric assay [S2]. The nanozymes (1 mg mL -1 , 100 µL) were added into the HAc-NaAc buffer (0.1 M, pH 3.0, 1 mL) containing H2O2 (1 M, 1 mL) and MB (1 mM, 100 µL), respectively. Then, the absorbance of the reaction solution was monitored after 1.5 h.

S2.7 Interference Study for AChE Detection
A series of proteins were chosen to measure the anti-interference ability of the NO2-MIL-101 based biosensor. Instead of AChE, 50 µL 10 µg mL -1 HRP (horseradish peroxidase), LAC (Laccase), GOx (Glucose oxidase), INV (Invertase), and BSA (bovine serum albumin) were added into the reaction system respectively. The absorbance values at 652 nm were recorded to evaluate the interference effect.

S2.8 Recovery Test for AChE
Human serum samples diluted 100 times for the recovery test of AChE. Then different concentration AChE were added into the diluted serum samples for the recovery tests.

S2.9 Colorimetric Detection of Paraoxon-ethyl
AChE (50 mU mL -1 , 50 µL, pH 7.4) and different concentration paraoxon-ethyl (50 µL) were incubated for 30 min 37 °C. The ATCh (10 mM, 30 µL, pH 7.4) was introduced into this system for another 30 min. The subsequent processes were the same as the procedure of AChE detection and the absorbance of this system was named as A1. Two parallel experiments were carried out at the same time. For one paraoxon-ethyl was not added into the reaction system (A). For another, the paraoxonethyl and ATCh were not added into the reaction system (A0). The inhibition rate was used to measure the amounts paraoxon-ethyl, and the inhibition rate was calculated by Eq. S6:

S2.10 Interference Study for Paraoxon-ethyl Detection
A series of interference substances were chosen to measure the anti-interference ability of the NO2-MIL-101 based biosensor. Instead of paraoxon-ethyl, 50 µL 1 µg mL -1 Na + , Ca 2+ , Mg 2+ , glucose and 50 µL 100 ng mL -1 avermectin, tebuconazole, chlorothalonil, fipronil were added into the reaction system respectively. The absorbance values at 652 nm were recorded to evaluate the interference effect.

S2.11 Recovery Test for Paraoxon-ethyl
Tap and river water samples were spiked with paraoxon-ethyl after a filtration. Besides, the rice and apple samples were washed with ultrapure water and then dried at room temperature. Then, 3 g of sample was placed into an ultrasonic bath for 5min before being centrifuged for 10 min (12,000 rpm). Later, a different amount of OP was added into the real samples. The concentration of the real samples was determined by the calibration curve.  The mass ratio was obtained by ICP-OES, and the atomic concentration was obtained by XPS.      Refs.