A simple and universal method for protein immobilization

Significance 

Numerous applications in analytical chemistry, bio-catalysis, biotechnology and in various medical devices are faced with an inherent problem relating to protein immobilization at interfaces. Basically, for successful application, the immobilization should not alter the protein conformation and maintain its bioactivity. The perfect method should also be unspecific with respect to the surface chemistry, surface geometry, and the protein to be immobilized. To this end, it is promising to adapt the so-called layer-by-layer (LbL) assembly of polyelectrolyte (PE), to the protein immobilization. This method consists in the alternate adsorption of oppositely charged PE, which results in a PE multilayer. By alternating the adsorption of a protein with a PE, it is, in theory, possible to construct a multilayer that immobilizes the protein. Nevertheless, integrating proteins in LbL thin films is very challenging due to their low conformational entropy, heterogeneous spatial distribution of charges, and polyampholyte nature.

Protein−polyelectrolyte complexes (PPCs) are promising building blocks for LbL construction owing to their standardized charge and polyelectrolyte corona. Unfortunately, the true advantage that they bring compared to bare protein integration have not been shown yet.

To this note, a team of researchers at UCLouvain led by Professor Christine Dupont-Gillain from the Institute of Condensed Matter and Nanosciences in Belgium demonstrated that the electrical charge of a protein could be standardized by a polyelectrolyte (PE) and that the resulting PPCs could be used to obtain sustainable multilayer growth. In particular, they showed that the multilayer construction using PPCs could be based on PE−PE interactions only and as such would allow the construction method to be generalized to all proteins. Their work is currently published in the research journal, ACS Nano.

In brief, their research team prepared PPCs through the complexation of lysozyme with low molar mass poly(styrene sulfonate) (PSS) in different conditions of pH and ionic strength. Next, the PPCs size and electrical properties were investigated, and the forces driving complexation were elucidated, in the light of computations of PE conformation, with a view to further unravel LbL construction mechanisms. Lastly, quartz crystal microbalance and atomic force microscopy were used to monitor the integration of PPCs compared to the one of bare protein molecules in LbL assemblies, and colorimetric assays were performed to determine the protein amount in the thin films.

The authors observed that the layers built with PPCs showed higher protein contents and hydration levels. In addition, the researchers noted that LbL construction with PPCs mainly relied on standard PE−PE interactions, i.e. independent of the charge state of the protein, in contrast to classical bare protein assembly with polyelectrolytes.

In summary, in the frame of the PhD thesis of Aurélien vander Straeten, the team of researchers presented the standardization of the electrical charge of a protein using polyelectrolyte, where the resulting PPCs could be used to obtain sustainable LbL growth. In general, it was seen that the balance between intramolecular electrostatic repulsion within PSS and electrostatic attraction between PSS and lysozyme was responsible for PPCs formation. Altogether, their observations considerably simplify the incorporation of proteins in multilayers, which will be beneficial for biosensing, heterogeneous bio-catalysis, biotechnologies, and medical applications that require active proteins to be immobilized on various surfaces.

A simple and universal method for protein immobilization - Advances in Engineering

About the author

In 2010, Aurélien vander Straeten began his studies in bioengineering at UCLouvain. In 2013, he chose to specialize in chemistry and bioindustries and, one year later, embarked on the nanobiotechnologies, materials and catalysis option. During his master’s degree, he carried out a month of research in oncology at the University of Maastricht, six months of study at KU Leuven, and a dissertation on vaccine formulations. In 2015, he graduated as a bioengineer and obtained a FRIA scholarship to finance his PhD in protein immobilization at interfaces. He is in the final year of his PhD.

About the author

Dr. Anna Bratek-Skicki obtained her doctorate in chemical science from the Institute of Catalysis and Surface Chemistry (ICSC) Poland. She was awarded a postdoctoral fellowship from the Irish Research Council for Science, Engineering and Technology and worked at the Centre for BioNano Interaction at University College Dublin. She returned to the ICSC and worked within a European Project (FUNANO) to develop functional nano-and microparticles for biotechnological applications. She was working as a Marie Curie Fellow at the Université catholique de Louvain, Belgium where her research involved the development of novel materials and structures for controlling protein adsorption.

Currently, she is working at the VIB-VUB Research Centre in Brussels where her research is focus on determining relationship between structure and function of intrinsically disordered proteins for developing novel drug(s) against neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer disease.

About the author

Charles-André Fustin got his Ph.D. in 1999 from the University of Namur (Belgium). From 2000 to 2001 he was a post-doctoral researcher at the Université catholique de Louvain (UCLouvain) in Belgium. He then moved to the group of Prof. H.W. Spiess in the Max-Planck Institute for Polymer Research in Mainz (Germany) where he got a European Marie-Curie Fellowship. In 2003 he obtained a Chargé de Recherches position of the FNRS at UCLouvain. Between 2007 and 2018 he was Research Associate then Senior Research Associate of the FRS-FNRS and since 2018 he is Professor of organic materials at UCLouvain.

His research interests include supra- and macromolecular chemistry, stimuli responsive polymers, polymer gels and networks, and mechanically-linked architectures.

About the author

Christine Dupont holds a bachelor’s degree in bioengineering (1995) and a PhD in agronomy and bioengineering (2000) from the UCLouvain Faculty of Bioengineering. After a postdoctoral fellowship at the University of Manchester (United Kingdom), she obtained a mandate as a postdoctoral researcher and then research associate (2005) of the National Fund for Scientific Research.

She is currently full professor at UCLouvain, where she teaches courses in the fields of chemistry, nanobiotechnologies and biomaterials, and leads an active research team in the field of biointerfaces and the mastery of interactions between living and non-living materials.

Reference

Aurelien vander Straeten, Anna Bratek-Skicki, ́ Alain M. Jonas, Charles-André Fustin, Christine Dupont-Gillain. Integrating Proteins in Layer-by-Layer Assemblies Independently of their Electrical Charge. ACS Nano 2018, volume 12, page 8372−8381.

Go To ACS Nano

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