Integrating Proteins in Layer-by-Layer Assemblies Independently of their Electrical Charge

Abstract

Layer-by-layer (LbL) assembly is an attractive method for protein immobilization at interfaces, a much wanted step for biotechnologies and biomedicine. Integrating proteins in LbL thin films is however 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 (PE) corona. In this work, lysozyme was complexed with poly(styrenesulfonate) (PSS) at different ionic strengths and pH values. The PPCs size and electrical properties were investigated, and the forces driving complexation were elucidated, in the light of computations of polyelectrolyte conformation, with a view to further unravel LbL construction mechanisms. 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. Layers built with PPCs show higher protein contents and hydration levels. Very importantly, the results also show that LbL construction with PPCs mainly relies on standard PE-PE interactions, independent of the charge state of the protein, in contrast to classical bare protein assembly with PEs. This considerably simplifies the incorporation of proteins in multilayers, which will be beneficial for biosensing, heterogeneous biocatalysis, biotechnologies, and medical applications that require active proteins at interfaces.

Voltammetric pH Measurements Using Azure A-Containing Layer-by-Layer Film Immobilized Electrodes

“…Formation of a thin multilayer film can be both driven by electrostatic interaction and by other types of interactions, such as biological affinity, e.g., avidin–biotin bonds [ 2 , 3 , 4 ], sugar–lectin bonds [ 5 ]; hydrogen bonds [ 6 , 7 ]; diol–phenylboronic acid bonds [ 8 , 9 ]; guest–host interactions [ 10 ]; and other low energy physical bonds [ 11 , 12 , 13 ]. Thus, a functional thin film can be formed from synthetic polymers and other materials, such as proteins, such as enzymes [ 14 , 15 ], polysaccharides [ 16 , 17 ], supramolecular compounds [ 18 ], and nanoparticles [ 19 ]. Furthermore, functional molecules can be easily immobilized in a film by modifying them with a polymer chain.…”

The Synthesis of a 2D Ultra‐Large Protein Supramolecular Nanofilm by Chemoselective Thiol–Disulfide Exchange and its Emergent Functions

“…This proteinaceous nanofilm presents robust interfacial adhesion with a wide range of substrates for universal surface modification due to its internal amyloid‐like adhesive structure and can controllably encapsulate and release functional molecules and colloids without resulting in a significant loss in activity of the encapsulated proteins. This approach thus not only offers a nontoxic strategy to prepare proteinaceous nanofilms or coatings on versatile material surfaces for surface modification/functionalization, which is important for cell controls, cell culture, and other biological applications, but also describes a strategy to immobilize and release active proteins on a surface without noticeable activity loss, which is a key challenge and has prime significance in the fields of biosensing, diagnostics, biomaterials science, and tissue engineering …”

Decomposition of Glucose-Sensitive Layer-by-Layer Films Using Hemin, DNA, and Glucose Oxidase

“…Various other interactions have also been recently employed to construct LbL films, such as hydrogen bonding [4,5] and sugar-lectin binding [6,7]. The materials employed for this purpose have included synthetic polymers [8,9], polysaccharides [10][11][12], protein [13][14][15], and DNA [16,17]. Such layered multilayer films have found application in separation and purification [18,19], sensors [20,21], and drug delivery systems (DDSs) [22][23][24].…”