Metal–organic framework (MOF) hybridized gold nanoparticles as a bifunctional nanozyme for glucose sensing

Inspired by natural enzymes that possess multiple catalytic activities, here we develop a bifunctional metal–organic frame-work (MOF) for biosensing applications. Ultrasmall gold nano-particles (AuNPs) are grown in the internal cavities of an iron (Fe) porphyrin-based MOF to produce a hybridized nanozyme, AuNPs@PCN-224(Fe), in which AuNPs and PCN-224(Fe) exhibit the catalytic activity of glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively. We established that the bifunctional nanozyme was capable of a cascade reaction to generate hydrogen peroxide in the presence of d-glucose and oxygen in situ, and subsequently activate a colorimetric or chemiluminescent substrate through HRP-mimicking catalytic activity. The nanozyme was selective over a range of other saccharides, and 93% of the catalytic activity was retained after being recycled five times.


S1. Experimental Section
Materials and Instrumentation.All purchased chemicals and reagents are of analytical grade.Tetra(4carboxyphenyl)porphine (TCPP), FeCl24H2O, ZrClO2•8H2O, chloroauric acid, 3,3ʹ,5,5ʹtetramethylbenzidine (TMB, 99%), hydrogen peroxide (H2O2, 30%), horseradish peroxidase (HRP), glucose oxidase (GOx), D-fructose, D-galactose, D-mannose, glucan (Dextran, Mw 100,000), and Dglucose were purchased from Sigma-Aldrich.Maltose, saccharose, lactose and beta-cyclodextrin were purchased from Aladdin.Pro-light HRP Chemiluminescent Kit was purchased from TIANGEN. 1 H NMR spectra were recorded on a Bruker AM 400 MHz spectrometer with tetramethylsilane (TMS) as the internal reference.High-resolution mass spectra (HRMS) were recorded with a Waters Micromass LCT mass spectrometer.Transmission electron microscopy (TEM, Talos F200X) or field-emission scanning electron microcopy (SEM, Helios G4 UC) was used to characterize the morphology of materials, and energy-dispersive X-ray (EDX) spectroscopy was used for elemental mapping.The particle size and Zeta potential of materials were obtained from a Zeta sizer Nano ZS (Malvern Instruments).Fourier transform infrared (FT-IR) spectra were measured on a Thermo Scientific Nicolet 6700.Powder X-ray Diffraction Spectroscopy (PXRD) was performed using Rotating Anode X-ray Powder Diffractometer (Rigaku, Japan).N2 sorption isotherms were collected using Tristar Ⅱ 3020 surface area/pore size analyzer.The surface composition and chemical states of materials were analyzed by X-ray Photoelectron Spectroscopy (XPS) performed on the ESCALAB 250Xi X-ray photoelectron spectrometer (Thermo Scientific, USA) using monochromatic Al Kα radiation.Electron Spin Resonance (ESR) spectra were performed on the EMX-8/2.7 ESR spectrometer.UV-vis absorption spectra were measured on a Varian Cary 500 UV-Vis spectrophotometer or a UV microplate reader (DR-3000).Chemiluminescence was measured on a Fiji LAS-4000 Super CCD Remote Control Science Imaging System.Chemiluminescence spectra were obtained with a Varian Cary Eclipse fluorescence spectrophotometer.
AuNPs were synthesized according to a previous report. 2In brief, HAuCl4 trihydrate (0.05 mmol) and PVP (20 mg) were mixed in 45 mL deionized H2O.After incubation for 10 min, a freshly-prepared NaBH4 solution (100 mM, 5 mL) was rapidly injected.The reaction mixture was then stirred at room temperature for 24 h to obtain AuNPs with an average size of 10 nm.
To construct the hybrid nanozyme, PCN-224(Fe) (10 mg) was dispersed in 10 mL of deionized H2O under sonication.Then, an aqueous solution of HAuCl4 (10 mM, 1 mL) was added dropwise into the vial under vigorous stirring.The vial was sealed and stirred vigorously for 12 h in the dark.Then, the resulting precipitates were collected by centrifugation (12000 rpm, 15 min) and washed twice with deionized H2O.The precipitates were redispersed into 10 mL of deionized H2O.Next, a freshly-prepared, icecold aqueous solution of NaBH4 (0.1 M, 250 L) was quickly injected followed by a vigorous stirring for another 30 min.Finally, AuNPs@PCN-224(Fe) were obtained as a fuchsia solid after centrifugation (12000 rpm, 15 min) and then washing with deionized H2O five times.
Electron spin resonance (ESR) experiment.5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was used as the spin trapping agent.In a typical measurement, 10 μL of AuNPs@PCN-224(Fe) aqueous suspension (final concentration 100 mg L -1 ) and 10 μL of 500 mM D-glucose were diluted with 380 μL of phosphate-buffered saline (PBS, 0.1 M, pH 7.4).Immediately, 100 μL of 100 mM DMPO was added into the above mixture.The resultant solution was extracted by quartz capillary tube and placed in a glass tube.After 5 min, the spectrum was recorded on a ESCALAB 250Xi.

Measuring the peroxidase-like activity of PCN-224(Fe).
To a PBS (0.1 M, pH 7.4) of PCN-224(Fe) (100 μg mL -1 ), TMB (5 mM, used as substrate) and H2O2 (5 mM) were added.After the mixture was incubated for 10 min, the absorption of TMB was measured by a Varian Cary 500 UV-Vis spectrophotometer or a UV microplate reader (DR-3000).The peroxidase activity of PCN-224 and HRP was measured by the same protocol.

Measuring the cascade catalytic activities of AuNPs@PCN-224(Fe).
To a PBS (0.1 M, pH 7.4) of AuNPs/PCN-224(Fe) (1 mg mL -1 ), D-glucose (0.2 M) and TMB (5.0 mM) were added.After the mixture was incubated for 40 min, The absorption of TMB was measured by a Varian Cary 500 UV-Vis spectrophotometer or a UV microplate reader (DR-3000).For the selectivity assay, D-fructose, maltose, saccharose, lactose, D-galactose, D-mannose, glucan (Dextran, Mw 100,000) and Beta-cyclodextrin (0.2 M) were used separately instead of D-glucose.The cascade catalytic activities of AuNPs, PCN-224(Fe), GOx, HRP, mixture of COx and HRP, and mixture of AuNPs and PCN-224(Fe) were measured by the same protocol.For the chemiluminescent assay, Luminol (1 mM) was used as the substrate instead of TMB, and the chemiluminescent signals were measured on a Fiji LAS-4000 Super CCD Remote Control Science Imaging System.
To test the recyclability of the bifunctional nanozyme, AuNPs/PCN-224(Fe) was recovered by centrifugation (12 000 rpm, 2 min) after one catalytic cycle (40 min) and its concentration was calibrated by UV-vis spectroscopy.This action was repeated five times.
A serum-like solution was prepared according to the literature 3,4 .A mixture of 0.14 M sodium chloride, 4.2 mM sodium bicarbonate, 3.0 mM potassium chloride, 1.5 mM magnesium chloride, 2.6 mM calcium chloride, 1.2 mM dibasic potassium phosphate, 0.5 mM sodium sulfate, 5 mM D-glucose, 5 mM cholesterol, 20 μM bilirubin, 3.0 mM of common blood plasma amino acids (glutamine, glycine, valine, arginine, lysine and alanine at equimolar concentrations of 0.5 mM) and 35 g L -1 human serum albumin was dissolved in deionized water.the pH of the resulting solution was adjusted to 7.40 with HCl (1.0 M).Then, test solutions were diluted in distilled water to ensure that the D-glucose concentrations were within the linear range of the calibration curve and to reduce possible matrix effects.Recoveries were also determined in the same samples by spiking the raw (undiluted) samples with different concentrations of D-glucose.Then, the final concentrations of D-glucose in the spiked samples were detected by the above colorimetric or chemiluminescent detection procedure.All tests were repeated five times.
Kinetics study.The reaction kinetics of the nanozymes was measured using TMB as the substrate.For the peroxidase-like catalytic reaction, PCN-224(Fe) (1 mg mL -1 ) was added into a PBS (0.1 M, pH 7.4) containing 10 mM H2O2 and TMB of different concentrations (0.0125, 0.025, 0.05, 0.1, 0.3, 0.5 and 0.8 mM).Then, the absorbance at 652 nm of the solutions was measured in time-course mode on a UV microplate reader (DR-3000).For the cascade reaction, AuNPs@PCN-224(Fe) (1 mg mL -1 ) was added into a PBS (0.1 M, pH 7.4) containing 5 mM TMB and D-glucose of different concentrations (0.05, 0.1, 0.3, 0.5, 1, 2 and 3 mM).Then, the absorbance at 655 nm of the solutions was measured in time-course mode on a UV microplate reader (DR-3000).Table S3.Comparison of the sensitivity of different colorimetric methods for D-glucose sensing.

Fig. S4 .
Fig. S4.Dark-field TEM image of AuNPs@PCN-224(Fe).The orange dots represent AuNPs, which are uniformly distributed in PCN-224(Fe) in general; however, particles with larger sizes could be seen on the surface of the MOFs.

Fig. S12 .
Fig. S12.(a) Plotting the absorption changes of TMB (1 mM) with different indicated concentrations of PCN-224(Fe) in the presence of H2O2 (0.1 mM) as a function of time.(b) Plotting the absorption changes of TMB (1 mM) with different indicated concentrations of H2O2 in the presence of PCN-224(Fe) (1 μg mL -1 ) as a function of time.A and A0 are the absorption of TMB in the absence and presence of analytes, respectively.Error bars mean standard deviation (n = 3).

Fig. S13 .
Fig. S13.Plotting the absorption changes of TMB (5 mM) with different indicated concentrations of Dglucose in the presence of Hybrid (1 mg mL -1 ) as a function of time.A and A0 are the absorption of TMB in the absence and presence of analytes, respectively.Error bars mean standard deviation (n = 3).

Fig. S14 .
Fig. S14.Measuring the peroxidase-like activity of PCN-224(Fe) and cascade catalytic activities of Hybrid by measuring the CL intensity of Luminol in PBS solution under different indicated conditions.The concentrations used for H2O2, Luminol, D-glucose, PCN-224(Fe) and Hybrid are 5 mM, 5 mM, 1 mM, 1 mg mL -1 , and 1 mg mL -1 , respectively.

Fig. S16 .
Fig. S16.(a) Particle size changes of Hybrid stored in PBS (pH 7.4) over time.(b) Catalytic activity changes of Hybrid stored in PBS (pH 7.4) at 4 °C and 25 °C.Measurement of the catalytic activity of Hybrid as a function of (c) pH, and (d) Temperature.Analysis of the catalytic activity of Hybrid by measurement of the UV-vis absorbance (at 652 nm) of TMB in PBS solution under the various indicated conditions.The concentrations used for Hybrid, TMB and D-glucose were 1 mg mL -1 , 5 mM and 0.2 mM, respectively.

Fig. S17 .
Fig. S17.Measuring the catalytic activity of AuNPs@PCN-224(Fe) after being recycled for one to five times.Note that the concentration of the nanozyme was calibrated before each test to avoid bias from simple quantity loss of the material during recovery.

Table S4 .
Comparison of the sensitivity of different chemiluminescent methods for D-glucose sensing.

Table S5 .
Recovery rate of D-glucose added to a serum-like solution determined by the colorimetric and chemiluminescent assays using AuNPs@PCN-224(Fe) as the catalyst.
a RSD = Relative standard deviation.