We used polymer brushes composed of combinations of: poly(ethylene oxide) (PEO), a well-known protein-repellent polymer, poly(acrylic acid) (PAA, Mn = 2000 g/mol), a polyanion, and poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, Mn = 8500 g/mol), a polycation. Both polyelectrolytes are weak and therefore bear a variable density of negative and positive charges depending on pH. A gold substrate was modified by these thiolated polymers according to the “grafting to” method, in which end-functionalized polymers react with the surface to form tethered chains. The properties of such mixed brushes (PEO/PAA, PEO/PDMAEMA) were adjusted by using different ratios of both polymers in grafting solutions, and different molar masses of PEO. The molar masses of PAA and PDMAEMA were kept constant. The polymer brushes were characterized by the following methods: Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D), X-ray Photoelectron Spectroscopy (XPS), Time-of-Flight Ion Mass Spectrometry (ToF-SIMS, Atomic Force Microscopy (AFM), Water Contact Angle measurements and Streaming Potential measurements. The polymer brushes were studied for their conformational changes (swelling/shrinking behavior), which were regulated by pH and ionic strength.
Four proteins, Human Serum Albumin (HSA), Human Fibrinogen (Fb), Lysozyme (Lys), and Avidine (Av) were chosen to study their adsorption on the mixed brushes. Protein adsorption was first monitored from a single protein solution on the different brushes, using QCM-D. ToF-SIMS was used in order to collect useful information for further study of protein adsorption from a mixture of proteins, i.e. to identify characteristic protein fragments. Conditions for effective adsorption and desorption of single proteins on the polymer brushes were also determined.
Protein adsorption from mixtures of proteins was then studied using QCM, ToF-SIMS coupled with PCA and gel electrophoresis with silver staining. The latter technique allows the detection of less than 10 ng of proteins, making it extremely useful for applications involving low protein levels. We have successfully demonstrated the effectiveness of the smart PEO/PAA brushes to selectively adsorb proteins from a mixture of HSA/Fb/Lys. The selected proteins can then be effectively removed in selected conditions. Therefore, the adsorption/desorption cycles can be repeated on the same brushes.
Selective protein adsorption on PEO/PDMAEMA brushes was studied from mixtures of HSA/Lys, HSA/Av, and HSA/Fb/Lys. It was confirmed that HSA can be selectively adsorbed on the mixed brushes from HSA/Lys, HSA/Av, HSA/Fb/Lys mixtures, and effectively desorbed in selected conditions.
