Target-triggered cascade assembly of a catalytic network as an artificial enzyme for highly efficient sensing

Abstract

Determining the catalytic activity of artificial enzymes is an ongoing challenge. In this work, we design a porphyrin-based enzymatic network through the target-triggered cascade assembly of catalytic nanoparticles. The nanoparticles are synthesized via the covalent binding of hemin to amino-coated gold nanoparticles and then the axial coordination of the Fe center with a dual-functional imidazole or pyridine derivative. The network, which is specifically formed by coordination polymerization triggered by Hg²⁺ as the target, shows high catalytic activity due to the triple amplification of enzymatic activity during the cascade assembly. The catalytic dynamics are comparable to those of natural horseradish peroxidase. The catalytic characteristics can be ultrasensitively regulated by the target, leading to a selective methodology for the analysis of sub-attomolar Hg²⁺. It has also been used for "signal-on" imaging of reactive oxygen species in living cells. This work provides a new avenue for the design of enzyme mimics, and a powerful biocatalyst with signal switching for the development of biosensing protocols.

Fig. 1. Schematic representation of the cascade assembly of high catalytic networks. (A) The synthesis of N-AuNPs using PADS as both a reducing agent and a protection ligand, and Au-Hem conjugates via an amide reaction. (B) The assembly of MPT on Au-Hem through axial interactions. (C) The preparation of an enzymatic network via Hg²⁺-triggered polymerization and the catalytic oxidation of tyramine by the network in the presence of ROS.

Fig. 2. (A) Photograph and (B) UV-vis spectra of AuNPs prepared with PADS and HAuCl₄ at mass ratios of 2.6, 3.0, 3.2, 5.0, 7.0, 11.0, 26.4, 55.4 and 142.4 from 1 to 9. (C) ³¹P NMR and (D) ¹H NMR spectra of PADS (red) and the reaction mixture at a mass ratio of 5.0 (blue).

Fig. 3. (A) The Fourier transform infrared spectra of N-AuNPs (black), hemin (blue) and Au-Hem (magenta). (B) The UV-vis spectra of N-AuNPs and Au-Hem (1 μM equivalent hemin) solutions. (C) The Fe 2p XPS spectra of hemin and the hemin–MPT complex. (D) The change in the UV-vis spectrum upon the titration of MPT to hemin (20 μM) in pH 7.4 Tris–HCl at 36 °C. (E) The UV-vis spectra of MPT (10 μM) in the presence of different metal ions (100 μM). (F) A Job’s plot for the binding of Hg²⁺ to MPT. The total concentration of MPT and Hg²⁺ was kept constant at 100 μM in pH 7.4 Tris–HCl.

Fig. 4. (A) DLS assays of Au-Hem-MPT (0.1 μM equivalent hemin) before and after the addition of Hg²⁺ (0.1 μM). (B) The TEM images of Au-Hem-MPT (0.1 μM equivalent hemin) in the absence (top) and presence (bottom) of Hg²⁺ (0.1 μM). The scale bars represent 50 nm.

Fig. 5. (A) Fluorescence intensities of the oxidation product of tyramine (0.14 mM) catalyzed by N-AuNPs (1.0 nM), hemin, Au-Hem, Au-Hem-MPT and Au-Hem-Net (10 nM equivalent hemin) in the presence of H₂O₂ (0.4 mM) at 37 °C. (B) The fluorescence responses of the oxidation product of tyramine (0.14 mM) by H₂O₂ (0.4 mM), which is catalyzed by the adduct of Au-Hem (10 nM equivalent hemin) and MPT at marked concentrations in the absence (black) and presence (red) of Hg²⁺ (1.0 nM) at 37 °C. (C) The fluorescence response of the oxidation product of tyramine (0.14 mM) by 0.4 mM H₂O₂ for 15 min at 37 °C in the presence of an incubated mixture of Au-Hem (10 nM equivalent hemin) and different concentrations of Hg²⁺. (D) The fluorescence intensities of the oxidation product of tyramine (0.14 mM) by H₂O₂ (0.4 mM), which is catalyzed by Au-Hem-MPT (10 nM equivalent hemin) in the presence of marked metal ions at 37 °C. The concentration of Hg²⁺ is 1.0 nM, and the concentrations of the other metal ions are 10 nM.

Fig. 6. (A) Time-dependent UV-vis spectra of MPT (100 μM) in the presence of Hg²⁺ (100 μM). (B) The time-dependent fluorescence intensity of the oxidation product of tyramine (0.14 mM) catalyzed by Au-Hem-Net (10 nM equivalent hemin) in the presence of 0.4 mM H₂O₂ at 37 °C. (C) The fluorescence spectra of the oxidation product of tyramine (0.14 mM) by H₂O₂ (0.4 mM), which is catalyzed by Au-Hem-MPT (10 nM equivalent hemin) in the presence of Hg²⁺ at marked concentrations at 37 °C. (D) A plot of F – F ₀ vs. the logarithmic value of Hg²⁺ concentration. F ₀ and F are the fluorescence intensity in the absence and presence of Hg²⁺, respectively.

Fig. 7. The fitting curve of the initial pyrogallol oxidation profile at marked pyrogallol concentrations (A, C and E) and a Lineweaver–Burk plot of the pyrogallol oxidation (B, D and F) catalyzed by the Au-Hem conjugate (A and B, 0.2 μM hemin equivalent), Au-Hem-PMPT (C and D, 0.2 μM hemin equivalent) and Au-Hem-pNet (E and F, 0.2 μM hemin equivalent) in the presence of 0.4 mM H₂O₂.

Fig. 8. (A) Fluorescence responses of o-phenylenediamine (0.14 mM) oxidized by different ROS for 15 min in the presence of Au-Hem-Net (10 nM equivalent hemin) at an excitation wavelength of 410 nm. (B) The viability of HeLa cells that were detected with MTT after treatment with 100 μL of culture medium containing o-phenylenediamine (0.14 mM) and different volumes of Au-Hem-Net (100 nM equivalent hemin) for 5 h at 37 °C. (C–E) Confocal fluorescence images of HeLa cells after incubation with o-phenylenediamine and Au-Hem-Net (C), o-phenylenediamine (D) and Au-Hem-Net (E) for different times at 37 °C. o-Phenylenediamine: 0.14 mM; Au-Hem-Net: 10 nM equivalent hemin. The scale bars represent 250 μm.