Periodic Reporting for period 1 - SELECT-A-PROTEIN (Mixed brushes of stimuli-responsive polymers for the selective adsorption of proteins)
Reporting period: 2015-10-01 to 2017-09-30
Protein adsorption at solid/liquid interfaces is a common phenomenon that plays a key role in many biomedical and biotechnological areas. It is a crucial event for the development of applications such as enzyme carriers, biosensors, drug delivery systems, immunological assays, cell culture substrates and so forth. On the other hand, non-specific protein adsorption on sensor surfaces or assay platforms is problematic because it degrades the analytical performances of the device. Therefore, the desire to predict, control, and manipulate protein adsorption on different surfaces has been the main driving force in this arena.
Many different strategies have recently been developed in order to control protein adsorption. Most of them are based on the use of polymers at interfaces. Protein adsorption may be efficiently tuned using smart interfaces based on stimuli-responsive brushes. These systems are very promising for the development of numerous applications due to their ability to switch interaction forces between the brush and the surrounding environment. Mixed polymer brushes, composed of a protein adsorbing and of a protein-repellent polymer, are of particular interest due to their ability to finely control protein adsorption. The brush properties can be controlled by the amount of both polymers, polymer chain length, and by applying external triggers such as pH, ionic strength, etc. Based on this concept, we set two specific objectives: 1) developing a surface layer for the temporal control of protein adsorption, and 2) achieve the selective adsorption of proteins from a mixture of proteins in a stimuli-triggered manner.
In this project, we built brushes using a combination of either a polyanion or a polycation with a protein-repellent polymer. The properties of these brushes were adjusted by different ratios of both polymers, different molar masses of the protein-repellent polymer, and ionic strength and pH changes in the surrounding medium.
We have successfully achieved the goal of this research project during the past 2 years, with the development of two systems that allows one to selectively adsorb one protein from a mixture of proteins and to further completely desorb it from the surface. This opens perspectives for protein separation, diagnostic applications, etc
Many different strategies have recently been developed in order to control protein adsorption. Most of them are based on the use of polymers at interfaces. Protein adsorption may be efficiently tuned using smart interfaces based on stimuli-responsive brushes. These systems are very promising for the development of numerous applications due to their ability to switch interaction forces between the brush and the surrounding environment. Mixed polymer brushes, composed of a protein adsorbing and of a protein-repellent polymer, are of particular interest due to their ability to finely control protein adsorption. The brush properties can be controlled by the amount of both polymers, polymer chain length, and by applying external triggers such as pH, ionic strength, etc. Based on this concept, we set two specific objectives: 1) developing a surface layer for the temporal control of protein adsorption, and 2) achieve the selective adsorption of proteins from a mixture of proteins in a stimuli-triggered manner.
In this project, we built brushes using a combination of either a polyanion or a polycation with a protein-repellent polymer. The properties of these brushes were adjusted by different ratios of both polymers, different molar masses of the protein-repellent polymer, and ionic strength and pH changes in the surrounding medium.
We have successfully achieved the goal of this research project during the past 2 years, with the development of two systems that allows one to selectively adsorb one protein from a mixture of proteins and to further completely desorb it from the surface. This opens perspectives for protein separation, diagnostic applications, etc
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.
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.
The mixed polymer brushes developed in SELECT-A-PROTEIN are novel, and allows one to selectively adsorb a given protein from a mixture of proteins. Since the four chosen proteins are different in size, shape and iep, the behavior of the mixed brushes can be applied for other biomolecules or their mixtures. These “smart surfaces” have potential applications such as controlled drug-delivery and release systems, as well as for sensing very small concentrations of analytes. The work has a great significance in the biomedical and pharmaceutical sectors. Society as a whole would benefit from the introduction of modern and sensitive systems in the biomedical and pharmaceutical fields.