Study On Highly Sensitive Immunoassay Based On Catecholamine Functional Materials

The ultra-sensitive and precise detection of protein biomarkers plays a vital role in clinical diagnosis and biomedical research.Therefore,the application and innovation of immunoassay methods have attracted widespread attention.Among them,colorimetric immunoassay has the advantages of simple operation,visualization,and fast reading speed,and is widely used in the determination of trace components.Catecholamine,as a neurotransmitter,is an amine compound containing catechol（ie catechol）,which is closely related to people’s health and disease.Because catecholamines contain amino and phenolic hydroxyl groups,they can be used to prepare functional materials with excellent physical and chemical properties through oxidation,complexation with metal ions or supramolecular interaction.Catecholamine functional materials are used in immunoassay methods,showing good reactivity and free radical scavenging ability,thereby effectively improving the sensitivity of detection.（1）In this work,the mussel-inspired polydopamine（PDA）coatings with biocompatible and reactive surface characteristics were desirably incorporated with the high loading capacity and p H-induced disassembly-releasing behaviors towards Fe（III）ions to develop an enzyme-free colorimetric immunoassay.Clustered ferroferric oxide nanoparticles（Fe₃O₄ NPs）with superpara magnetism were simultaneously coated with PDA layers and Fe（III）ions to fabricate the Fe₃O₄@PDA-Fe（III）composites as the label of target analyte,in which Fe₃O₄ NPs with fine peroxidase-like activity can simplify the labeling process via facile magnetic separation.Notably,the loaded Fe（III）ions can be easily released into the solution by the p H-induced disassembly property of PDA layers,thus making it efficient to in situ generate prussian blue（PB）NPs by the reaction between the released Fe（III）ions and ferrocyanide.Based on the high peroxidase-like activity of PB NPs,excellent synergetic catalytic effect between PB and Fe₃O₄NPs was triggered during the colorimetric reaction,which significantly improved the detection sensitivity when using prostate specific antigen（PSA）as the target analyte.Moreover,favorable linearity was found in the range of 0.0005-20 ng·m L^-1with a low detection limit of 0.84 pg·m L^-1（S/N=3）,which offers a facile and reliable method for the sensitive detection of PSA and other cancer biomarkers in human serum.（2）Inspired by the first working idea,the catechol functional network of PDA is directly used as a scaffold to cooperate with Fe（III）ions to form PDA-Fe（III）NPs to construct a colorimetric immunosensor,which significantly improves the sensitivity of detection.In details,PDA-Fe（III）NPs can maintain in a disassembled tri-coordinate state under alkaline conditions while the highly-loaded Fe（III）ions can be easily released under acid conditions to react with ferrocyanide for the in situ generation of prussian blue（PB）NPs.This may be due to the higher Fe（III）ions content of PDA-Fe（III）NPs and the generation of more PB to participate in colorimetric reaction,while Fe₃O₄@PDA-Fe（III）NPs is just a thin shell,forming less PB with catalytic properties,and the exposed Fe₃O₄ does not contribute to higher catalytic effect,which improves the detection sensitivity of PSA.In addition,favorable linearity was found in the range of 0.0005-20 ng·m L^-1with a low detection limit of 0.84 pg·m L^-1（S/N=3）.（3）In this work,we constructed a novel colorimetric-fluorescence dual-mode immunosensing strategy based on Epinephrine（Ep）for the accurate and quantitative determination of cytokeratin 19 fragment 21-1（CYFRA 21-1）.Ep,induced an allochroic reaction,is catalytically oxidized by Ni Pd NPs with excellent activity and durability in the presence of H₂O₂.Then,the oxidation product adrenochrome undergoes tautomerism under alkaline conditions to produce adrenolutin with yellow-green fluorescence characteristics.The colorimetric and fluorescent signals can generate from a same sample and the consequent dual-mode readout can be performed on a single instrument,which reduce the batch effects.Under the optimal condition,the sensing strategy exhibited a linear range from 0.0005 to 20 ng·m L^-1,and the detection limits were 36.1 and 42.0 fg·m L^-1（S/N=3）for the colorimetric and fluorescent modes,respectively.