Multivariate Analysis On The Association Kinetic Model Of Protein- Nanoparticle Interaction

Association kinetics of proteins on a substrate surface are major concerns in biology,medicine,biotechnology,and food processing.Generally,time-resolved spectroscopy can be used to monitor the proteinnanoparticle adsorption process.However,due to the severe overlapping in many kinetic data,it is difficult to distinguish components with a traditional univariate analysis method.Thus,it is essential to introduce the multivariate analysis method to conduct the kinetic analysis of complex system.This dissertation consists of two case-studies for modeling the protein-nanoparticle interaction.In the first case,a semi-quantitative investigation was designed to compare the kinetic processes of two highly homologous proteins,i.e.human and bovine serum albumim(HSA and BSA,respectively),adsorbing onto the surface of gold nanoparticles(AuNPs)by combining UV-vis spectroscopy,chemometrics technique,and mathematical modelling method.Furthermore,a series of equations with multiple exponential functions were tested to portray the dynamic conversion of the original surface(i.e.citrate-capped nanoparticles)towards the modified surface(i.e.protein-conjugated nanoparticles).We found that the adsorption of serum albumin on gold nanoparticle was best deciphered as the two-mode competitive kinetics.Interestingly,for HSA the slower mode contributed considerably to the localized surface plasmon resonance response of protein-conjugated AuNPs,while in the case of bovine serum albumin the faster mode overwhelmed the slower one at the high protein concentrations.The kinetic patterns were rationalized with the diversity of biomolecular orientations on the nanoparticle surface and protein features.As one of following-up works,we further carried out the kinetic investigation of a fiber-forming functional protein,REG3 A,adsorbing on the citrate-capped gold nanoparticle with the similar protocol.The conjugation of REG3 A on AuNPs itself is a relatively more complex process.Unlike globulin protein wrapping on the surface of gold in the way of single layer,REG3 A has very strong self-aggregation tendency to form fiber network.As long as the assembly occurs,aggregated REG3 A will fall off from the nanosurface to environment,presenting a multi-process behavior of repeated fluctuation in the kinetic model.The aggregationexfoliation mechanism was verified by atomic force microscopy(AFM)and stability study.In the presence of lipopolysaccharide(LPS),the small molecule will occupy the surface of nanoparticle before REG3 A absorbing.When the LPS concentration increased to 10 ?g/mL,the intervention effect on REG3 A adsorption towards the surface of AuNPs was observed,significantly decreasing the component number of the kinetic model and smoothing the absorption curves as well.The small-molecule effect on protein conjugation might provide a new solution of controlling protein modification on nanosurface.In summary,this dissertation work achieved the methodology innovation on the two-dimensional data analysis with the combination of the multivariate analysis and association kinetics,successfully unveiling absorption patterns and mechanisms of protein-nano interaction.The simple but efficient protocol is potential to frabricate nano-based biosensor for analyzing the kinetic behaviors of low-expressed proteins,such as in the exploration of disease-relavant proteins or enzyme molecules.