In Situ Surface Plasmon Resonance Studies On Polydopamine Thin Film-protein Interactions

Adsorption/desorption of protein on a solid surface is a basic step of many essential life processes such as cell adhesion,signal transduction,tissue development and so on.The study of protein-surface interaction is not only fundamentally important,but also may provides invaluable guidance for various applications including medical diagnostic materials,biochip,cell culture,drug delivery,etc.The adsorption of protein on a solid surface is a complex process involving various factors.Including the physicochemical properties of both protein and surface,and the surrounding environment.Regulating the chemical composition of solid surfaces to control the protein adsorption is one of focuses receiving intensive research in recent years.Mussel-inspired polydopamine(PDA)is a multi-functional polymer coating with good biocompatibility,hydrophilicity,adhesive ability and chemical activity.In recent years,PDA film has widely used in biosensing,drug delivery,biomedical imaging,tissue engineering and other fields.Due to the presence of functional groups such as catechol and quinone,PDA film can covalently bind with the proteins via Michael addition or Schiff's base formation,thus offering a universal surface modification layer for protein immobilization.Despite the essential importance of PDA-protein interaction,our knowledges remains limited so far regarding,for example,the immobilization density of protein on PDA,the kinetics and thermodynamics of protein-PDA reaction,and the stability of attached protein on PDA surface,all of which are critical for both fundamental and practical research.The situation is further complicated if considering the following three facts.1)The oxidative polymerization mechanism of PDA is elusive at this time due to the complex redox processes involved and a series of intermediates generated during the reaction.The exact chemical components of PDA are still under debate and therefore it is hard to figure out a clear picture about the protein-PDA interaction;2)A number of methods have been established to grow PDA films(different buffer,oxidant,polymerization duration,buffer pH,etc.);the film thickness,roughness,and chemical components may significantly vary with the synthetic conditions;3)the chemical components in the PDA film may also vary upon the environment change such as pH value due to the disturbed equilibriums of several reactive component pairs.Therefore,it is particularly of significance to investigate the protein-PDA interaction with a real-time and in situ manner.On the other hand,despite the unique advantages as multi-functional surface modified layers,PDA thin film's protein immobilization capacity is not very high when compared to other reactive surfaces with epoxy,carboxyl-derived NHS ester or amine groups,which limits its widespread applications.It would be very interesting if certain method could be developed to modulate the chemical component of PDA to improve its protein loading capacity.In this work,a systematic study was carried out to solve above problems mainly by in situ and real time Surface Plasmon Resonance(SPR)technique.The interactions between protein and PDA film have been investigated in detail and a novel strategy was developed to enhance the protein immobilization capacity of PDA film.The work has two major components:(1)The protein-PDA interaction,including protein adsorption behavior on PDA film,the kinetics of protein-PDA interaction and the effects of external factors(growth medium and pH)on protein adsorption on PDA was systematically studied by using SPR coupled to fluorescent protein microarray technology.The unique fluorescence quenching property of PDA film was disclosed and the quenching efficiency varies upon the change of PDA growth conditions(growth medium,growth pH and growth time).This part work may help us clarify the elusive details on protein-PDA interaction,and offer a useful guide for PDA's application involved biomolecules.(2)PDA thin film was modulated by electrochemical means with the purpose of enhancing its protein attachment capacity.The core idea is to electrochemically shift the catechol/quinone equilibrium on PDA thin film to improve the surface density of active quinone group and in turn the protein immobilization capacity.SPR was used to monitor the PDA growth as well as to quantitatively evaluate the immobilization amount of protein on PDA surfaces before and after the electrochemical manipulation.Quantitative investigations confirm that proper electrochemical modulation could improve the protein loading capacity for up to 27%.In addition,spectroscopic investigations were conducted and the results confirm the underlying mechanism of catechol/quinone equilibrium shift.This work demonstrates the feasibility to electrochemically enhance the protein attachment on PDA film and may provide a valuable guide to facilitate its various biorelated applications.