Controlled Synthesis Of Spherical Poly (Acrylic Acid) Brushes And Their Application In Biodetection

Polymer brushes form when one end of polymer was densely attached to spherical carrier such as nanoparticles. The merging of polymer science and surface science gives birth to this novel material, which arouses extensive interests among researches in this field owing to its various composition, well-defined structure, and distinctive surface properties. On the basis of previous researches, this thesis deals with scientific and application questions centered with spherical poly(acrylic acid) brushes(SPAABs). The thesis was composed of three major parts, namely, materials synthesis and characterization, interaction with protein, and application in biodetection.In the first part, we achieved the facile controlled synthesis and comprehensive characterization of SPAABs. A novel surface-initiated RAFT polymerization(SI-RAFT) route was designed to synthesize SPAABs. Compared with traditional SI-RAFT route that is usually complex and demanding, the proposed route greatly simplified the synthesis procedure by synthesizing a new RAFT chain transfer agent(RAFT CTA) functionalized with a silane group. Key structural parameters of SPAABs were determined by a combinational use of various characterization methods and the precise tailoring of materials structure was achieved. Conformation of SPAABs in aqueous solution and their response behavior were also studied by dynamic light scattering(DLS). Additionally, a UV-Vis spectrometry-based fitting method was proposed, which took advantage of the strong characteristic absorption of RAFT CTA to determine several key parameters including immobilization density of RAFT CTA, grafting density of active polymer chain, molecular weight of poly(acrylic acid)(PAA), re-initiation efficiency, and protein binding capacity. The controlled synthesis and in-depth characterization of SPAABs laid a solid foundation for the fundamental study of interaction between SPAABs and protein.In the second part, we illuminated a new mechanism and correspondingly developed a new method regarding the covalent immobilization of protein on SPAABs. Protein immobilization via the classical N-hydroxysuccinimide / N-(3-dimethyl-aminopropyl)-N’-ethyl- carbodiimidehydrochloride(NHS / EDC) coupling chemistry was studied. The hydrolysis kinetics of NHS ester and kinetics of protein immobilization in different pH were investigated and compared with conventional carboxylated silica particles, which revealed the governing role of electrostatic interaction in covalent immobilization process. This led to a further illustration on the competitive relation between electrostatic interaction and chemical reaction in different conditions and their influence on protein binding capacity. Based on the mechanism study, a new “chemical conjugation after electrostatic entrapment”(CCEE) method was developed, which comprised a tandem “electrostatic entrapment” and “chemical conjugation” step. By taking full advantage of unique “Donnan effect” of SPAABs, the CCEE method achieved high protein binding capacity while overcame the instability of electrostatic adsorption. Study on the mechanism and method of protein covalent immobilization on SPAABs paved the way for the application of SPAABs in biodetection to fully fulfill their superb properties as efficient protein carriers.In the third part, we developed a novel ultrasensitive enzyme-linked immunosorbent assay(ELISA) system using SPAABs as labels. Based on the preceding researches, the inner spaces and periphery of SPAABs were functionalized with horseradish peroxidase(HRP) and antibody via CCEE method and NHS / EDC process respectively, which endowed SPAABs with dual functionalities of effective analyte recognition and significant signal amplification. The amplification of SPAAB labels was much higher than conventional particles labels by virtue of high enzyme binding capacity and reservation of enzyme activity after immobilization. Using human Chorionic Gonadotrophin(hCG) as a model analyte, the SPAABs-amplified system achieved 267-fold improvement in detection sensitivity compared with conventional ELISA, which held great promise in early in vitro disease diagnosis.