Research On Construction Of New Iron Containing Nanozymes And Their Bioanalytical Applications

Inorganic nanomaterials are generally considered to be biologically inert,but surprisingly,the latest research reveals that many inorganic nanomaterials and even organic-inorganic hybrid nanomaterials possess the catalytic activities of natural enzymes.Such emerging nanomaterials that can mimic enzyme activity are called nanozymes.At present,nanozymes have become a new research hotspot in the field of enzymology since they have the advantages of high stability,low cost and easily regulated activity.The iron-containing nanozymes exhibit particularly high catalytic activity and stability,and stand out among the currently developed nanozymes.However,they still face a series of problems and challenges of the sensitivity to be improved,the detection mode to be enriched,and the detection range to be expanded in bioanalysis applications.We developed several new types of iron-containing nanozymes,and systematically studied their catalytic properties and catalytic mechanism of mimicking peroxidase.Furthermore,by combining the physicochemical properties and catalytic activity of these nanozymes,the highly sensitive sensing platforms for the detection of small biomolecules and cancer cells were constructed.Ferrous(II)phosphate nanoflowers(Fe₃(PO₄)₂·8H₂O NFs)were prepared by a facile co-precipitation method and were demonstrated to have peroxidase-like activity.The influencing factors of the catalytic activity of Fe₃(PO₄)₂·8H₂O and its catalytic kinetics were studied.The results showed that the catalytic activity was dependent on temperature,pH,and the concentrations of H₂O₂ and Fe₃(PO₄)₂·8H₂O.The kinetic study showed that Fe₃(PO₄)₂·8H₂O had a stronger affinity and higher catalytic efficiency towards H₂O₂ and 3,3?,5,5?-tetramethylbenzidine(TMB)than that of horseradish peroxidase(HRP).Study on the catalytic mechanism proved that its catalytic activity was derived from the generated hydroxyl radicals(·OH).Fe₃(PO₄)₂·8H₂O was further integrated with glucose oxidase(GOx)through a mild synthesis strategy.The GOx-Fe₃(PO₄)₂·8H₂O hybrid nanoflowers(HNFs)with hydrangea-like morphology were prepared.The catalytic activity and stability of the integrated nanozyme were investigated in detail.This integrated nanozyme displayed significantly enhanced catalytic activity and stability compared with free enzymes.On the basis of this integrated nanozyme with excellent catalytic performance,a sensing strategy for self-activated cascade detection of glucose was proposed.This sensing platform was successfully used to detect glucose in human serum.The linear range of glucose was as broad as 0.01–20 mmol/L,and the detection limit(LOD)was as low as 0.1?mol/L.The interference test proved that the method had high selectivity for glucose.The spike recovery test displayed that the recovery of glucose was 96.41%–108.29%and the RSD was less than 6.89%.A novel portable visual device for on-site detetction of glucose was designed based on the HNFs.A new multifunctional Fe₃(PO₄)₂·8H₂O-carbon dots(CDs)-folic acid(FA)hybrid nanoflowers(h NFs)with excellent peroxidase-like activity,fluorescent properties and specific targeting ability towards cancer cells was synthesized.The catalytic kinetics was investigated,and the result showed that this fluorescent nanozyme had a stronger affinity towards the double substrates in comparison with HRP and other reported nanozymes.Based on the excellent catalytic activity and the ability to target cancer cells of h NFs,a colorimetric and visual detection strategy of cancer cells was developed.This proposed strategy could distinguish as low as 25He La cells by the naked eye.The catalytic mechanism was further discussed,and it was confirmed that the h NFs could catalyze the converse of·OH from H₂O₂.Considering of this,the h NFs were employed for H₂O₂-and ascorbic acid(AA)-mediated the selective killing of cancer cells.The specific targeting ability of h NFs and therapeutic effects of the developed therapeutic strategies to cancer cells were further confirmed by aid of the fluorescent imaging ability of h NFs.As the terephthalic acid(TA)as the organic ligand,MIL-101(Fe)with octahedral morphology was prepared by a solvothermal method,and the catalytic properties and catalytic mechanism of MIL-101(Fe)as peroxidase mimics were studied in detail.It was displayed that MIL-101(Fe)had excellent peroxidase-like activity.Kinetic analysis displayed that a much stronger affinity of MIL-101(Fe)towards the two substrates than HRP.The study on catalytic mechanism demonstrated that the peroxidase-like activity of MIL-101(Fe)originated from the production of·OH.Since the organic ligand TA of MIL-101(Fe)could be oxidized by·OH to form a highly fluorescent product,MIL-101(Fe)was endowed with dual functions of mimetic peroxidase and fluorescent emission.A label-free fluorescence biosensor was successfully constructed for detection of choline in milk and acetylcholine(ACh)in human plasma by this bifunctional MIL-101(Fe)nanozyme.The linear ranges of choline and ACh were 0.1–10?mol/L and 0.01–100?mol/L,and the LOD were 20.0 nmol/L and 8.9 nmol/L,respectively.The interference test showed that the developed biosensor had high selectivity upon choline and ACh.The measured results of the actual samples by this method were consistent with the actual spiked choline concentrations and the ACh concentrations measured by a commercial assay kit.The recoveries of choline and ACh were in the range of99.63%–102.00%and 97.20%–102.91%,and the RSD were 1.02%–3.24%and0.88%–2.56%,respectively.These results suggested that the developed method exhibited high accuracy.A well-defined core-shell-shell magnetic microsphere with enhanced peroxidase-like activity was successfully synthesized by using Fe₃O₄ as the core,polydopamine(PDA)as the inner shell and MIL-101(Fe)as the outer shell.A discussion of the catalytic mechanism showed that the catalytic activity of Fe₃O₄@PDA@MIL-101(Fe)was attributed to the generation of·OH.The generated·OH caused the fluorescence enhancement of Fe₃O₄@PDA@MIL-101(Fe),while oxidizing the colorless o-phenylenediamine(OPD)to a yellow product(ox OPD),which in turn quenched the enhanced fluorescence.Given this,a novel fluorescent/colorimetric dual-readout strategy was built for the detection of choline in milk and ACh in human plasma.The linear range of choline was 0.05–40?mol/L with the LOD of 11.3 nmol/L by fluorescence method,while the linear range of choline was 0.05–50?mol/L with the LOD of 18.5 nmol/L by colorimetric method.The linear range of ACh was 0.02–20?mol/L with the LOD of 9.8 nmol/L by fluorescence method,while the linear range of ACh was 0.05–20?mol/L with the LOD of 16.6 nmol/L by colorimetric method.The measured results of the dual-readout sensing platform were in agreement with the actual spiked choline concentrations and the ACh concentrations measured by a commercial assay kit.The recovery of choline varied from 97.07%to 101.13%with the RSD ranging from0.55%to 1.83%,while the recovery of ACh varied from 97.52%to 103.55%with the RSD ranging from 0.98%to 3.28%,indicating that this sensing platform had high accuracy upon choline and ACh.