Dynamics of leaching of POPs and additives from plastic in a Procellariiform gastric model: Diet- and polymer-dependent effects and implications for long-term exposure.

Procellariiform seabirds are known to have high rates of plastic ingestion. We investigated the bioaccessibility of plastic-associated chemicals [plastic additives and sorbed persistent organic pollutants (POPs)] leached from plastic over time using an in vitro Procellariiform gastric model. High-density polyethylene (HDPE) and polyvinyl chloride (PVC), commonly ingested by Procellariiform seabirds, were manufactured with one additive [decabrominated diphenyl ether (PBDE-209) or bisphenol S (BPS)]. HDPE and PVC added with PBDE-209 were additionally incubated in salt water with 2,4,4'-trichloro-1,1'-biphenyl (PCB-28) and 2,2',3,4,4',5'-hexachlorobiphenyl (PCB-138) to simulate sorption of POPs on plastic in the marine environment. Our results indicate that the type of plastic (nature of polymer and additive), presence of food (i.e., lipids and proteins) and gastric secretions (i.e., pepsin) influence the leaching of chemicals in a seabird. In addition, 100% of the sorbed POPs were leached from the plastic within 100 hours, while only 2-5% of the additives were leached from the matrix within 100 hours, suggesting that the remaining 95% of the additives could continue to be leached. Overall, our study illustrates how plastic type, diet and plastic retention time can influence a Procellariform's exposure risk to plastic-associated chemicals.

Plasma membrane nanodeformations promote actin polymerization through CIP4/CDC42 recruitment and regulate type II IFN signaling.

In their environment, cells must cope with mechanical stresses constantly. Among these, nanoscale deformations of plasma membrane induced by substrate nanotopography are now largely accepted as a biophysical stimulus influencing cell behavior and function. However, the mechanotransduction cascades involved and their precise molecular effects on cellular physiology are still poorly understood. Here, using homemade fluorescent nanostructured cell culture surfaces, we explored the role of Bin/Amphiphysin/Rvs (BAR) domain proteins as mechanosensors of plasma membrane geometry. Our data reveal that distinct subsets of BAR proteins bind to plasma membrane deformations in a membrane curvature radius-dependent manner. Furthermore, we show that membrane curvature promotes the formation of dynamic actin structures mediated by the Rho GTPase CDC42, the F-BAR protein CIP4, and the presence of PI(4,5)P2. In addition, these actin-enriched nanodomains can serve as platforms to regulate receptor signaling as they appear to contain interferon-γ receptor (IFNγ-R) and to lead to the partial inhibition of IFNγ-induced JAK/STAT signaling.

Improvement of Lipid Detection in Mouse Brain and Human Uterine Tissue Sections Using In Situ Matrix Enhanced Secondary Ion Mass Spectrometry.

The potential of mass spectrometry imaging, and especially ToF-SIMS 2D and 3D imaging, for submicrometer-scale, label-free molecular localization in biological tissues is undisputable. Nevertheless, sensitivity issues remain, especially when one wants to achieve the best lateral and vertical (nanometer-scale) resolution. In this study, the interest of in situ matrix transfer for tissue analysis with cluster ion beams (Bin+, Arn+) is explored in detail, using a series of six low molecular weight acidic (MALDI) matrices. After estimating the sensitivity enhancements for phosphatidylcholine (PC), an abundant lipid type present in almost any kind of cell membrane, the most promising matrices were softly transferred in situ on mouse brain and human uterine tissue samples using a 10 keV Ar3000+ cluster beam. Signal enhancements up to 1 order of magnitude for intact lipid signals were observed in both tissues under Bi5+ and Ar3000+ bombardment. The main findings of this study lie in the in-depth characterization of uterine tissue samples, the demonstration that the transferred matrices also improve signal efficiency in the negative ion polarity and that they perform as well when using Bin+ and Arn+ primary ions for analysis and imaging.

Comparing the incidence of SARS-CoV-2 across age groups considering sampling biases - use of testing data of autumn 2021 in Belgium.

BACKGROUND: To design efficient mitigation measures against COVID-19, understanding the transmission dynamics between different age groups was crucial. The role of children in the pandemic has been intensely debated and involves both scientific and ethical questions. To design efficient age-targeted non-pharmaceutical interventions (NPI), a good view of the incidence of the different age groups was needed. However, using Belgian testing data to infer real incidence (RI) from observed incidence (OI) or positivity ratio (PR) was not trivial. METHODS: Based on Belgian testing data collected during the Delta wave of Autumn 2021, we compared the use of different estimators of RI and analyzed their effect on comparisons between age groups. RESULTS: We found that the RI estimator's choice strongly influences the comparison between age groups. CONCLUSION: The widespread implementation of testing campaigns using representative population samples could help to avoid pitfalls related to the current testing strategy in Belgium and worldwide. This approach would also allow a better comparison of the data from different countries while reducing biases arising from the specificities of each surveillance system.

Gas Cluster Ion Beams as a Versatile Soft-Landing Tool for the Controlled Construction of Thin (Bio)Films.

Surface biofunctionalization with proteins is the key to many biomedical applications. In this study, a solvent-free method for the controlled construction of protein thin films is reported. Using large argon gas cluster ion beams, proteins are sputtered from a target (a pool of pure proteins), and collected on a chosen substrate, being nearly any solid material. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed the presence of intact protein molecules on the collectors. Furthermore, lowering the energy per atom in the cluster projectiles down to 1 eV/atom allowed more than 60% of bradykinin molecules to be transferred intact. This protein deposition method offers a precise control of the film thickness as the transferred protein quantity is proportional to the argon clusters ion dose reached for the transfer. This major feature enables building protein films from (sub)mono- to multilayers, without upper limitation of the thickness. A procedure was developed to measure the film thickness in situ the ToF-SIMS instrument. The versatility and potential of this soft-landing alternative for further applications is demonstrated on the one hand by building a protein thin film at the surface of paper, a substrate hardly compatible with solution-based adsorption methods. On the other hand, the possibility to achieve alternated multilayer buildup is demonstrated with the construction of a bilayer composed of bradykinin and Irganox, with the two layers well separated. These results lay the first stone toward original and complex multilayers that could previously not be considered with solution-based adsorption methods, and this regardless of the substrate nature.

Layer-by-Layer Nanoarchitectonics Using Protein-Polyelectrolyte Complexes toward a Generalizable Tool for Protein Surface Immobilization.

Layer-by-layer (LbL) self-assembly is an attractive method for the immobilization of macromolecules at interfaces. Integrating proteins in LbL thin films is however challenging due to their polyampholyte nature. Recently, we developed a method to integrate lysozyme into multilayers using protein-polyelectrolytes complexes (PPCs). In this work, we extended this method to a wide range of protein-polyelectrolyte combinations. We demonstrated the robustness and versatility of PPCs as building blocks. LL-37, insulin, lysozyme, and glucose oxidase were complexed with alginate, poly(styrenesulfonate), heparin, and poly(allylamine hydrochloride). The resulting PPCs were then LbL self-assembled with chitosan, PAH, and heparin. We demonstrated that multilayers built with PPCs are thicker compared to the LbL self-assembly of bare protein molecules. This is attributed to the higher mass of protein in the multilayers and/or the more hydrated state of the assemblies. PPCs enabled the self-assembly of proteins that could otherwise not be LbL assembled with a PE or with another protein. Furthermore, the results also show that LbL with PPCs enabled the construction of multilayers combining different proteins, highlighting the formation of multifunctional films. Importantly, we show that the adsorption behavior and thus the multilayer growth strongly depend on the nature of the protein and polyelectrolyte used. In this work, we elaborated a rationale to help and guide the use of PPCs for protein LbL assembly. It will therefore be beneficial to the many scientific communities willing to modify interfaces with hard-to-immobilize proteins and peptides.

Magnesium rescues the morphology of Bacillus subtilis mreB mutants through its inhibitory effect on peptidoglycan hydrolases.

Cell wall homeostasis in bacteria is tightly regulated by balanced synthesis and degradation of peptidoglycan (PG), allowing cells to expand their sacculus during growth while maintaining physical integrity. In rod-shaped bacteria, actin-like MreB proteins are key players of the PG elongation machinery known as the Rod complex. In the Gram-positive model bacterium Bacillus subtilis depletion of the essential MreB leads to loss of rod shape and cell lysis. However, millimolar concentrations of magnesium in the growth medium rescue the viability and morphological defects of mreB mutants by an unknown mechanism. Here, we used a combination of cytological, biochemical and biophysical approaches to investigate the cell surface properties of mreB null mutant cells and the interactions of Mg2+ with the cell wall of B. subtilis. We show that ∆mreB cells have rougher and softer surfaces, and changes in PG composition indicative of increased DL- and DD-endopeptidase activities as well as increased deacetylation of the sugar moieties. Increase in DL-endopeptidase activity is mitigated by excess Mg2+ while DD-endopeptidase activity remains high. Visualization of PG degradation in pulse-chase experiments showed anisotropic PG hydrolase activity along the sidewalls of ∆mreB cells, in particular at the sites of increased cell width and bulging, while PG synthesis remained isotropic. Overall, our data support a model in which divalent cations maintain rod shape in ∆mreB cells by inhibiting PG hydrolases, possibly through the formation of crosslinks with carboxyl groups of the PG meshwork that affect the capacity of PG hydrolases to act on their substrate.

Embedding Collagen in Multilayers for Enzyme-Assisted Mineralization: A Promising Way to Direct Crystallization in Confinement.

The biogenic calcium phosphate (CaP) crystallization is a process that offers elegant materials design strategies to achieve bioactive and biomechanical challenges. Indeed, many biomimetic approaches have been developed for this process in order to produce mineralized structures with controlled crystallinity and shape. Herein, we propose an advanced biomimetic approach for the design of ordered hybrid mineralized nano-objects with highly anisotropic features. For this purpose, we explore the combination of three key concepts in biomineralization that provide a unique environment to control CaP nucleation and growth: (i) self-assembly and self-organization of biomacromolecules, (ii) enzymatic heterogeneous catalysis, and (iii) mineralization in confinement. We use track-etched templates that display a high density of aligned monodisperse pores so that each nanopore may serve as a miniaturized mineralization bioreactor. We enhance the control of the crystallization in these systems by coassembling type I collagen and enzymes within the nanopores, which allows us to tune the main characteristics of the mineralized nano-objects. Indeed, the synergy between the gradual release of one of the mineral ion precursors by the enzyme and the role of the collagen in the regulation of the mineralization allowed to control their morphology, chemical composition, crystal phase, and mechanical stability. Moreover, we provide clear insight into the prominent role of collagen in the mineralization process in confinement. In the absence of collagen, the fraction of crystalline nano-objects increases to the detriment of amorphous ones when increasing the degree of confinement. By contrast, the presence of collagen-based multilayers disturbs the influence of confinement on the mineralization: platelet-like crystalline hydroxyapatite form, independently of the degree of confinement. This suggests that the incorporation of collagen is an efficient way to supplement the lack of confinement while reinforcing mechanical stability to the highly anisotropic materials. From a bioengineering perspective, this biomineralization-inspired approach opens up new horizons for the design of anisotropic mineralized nano-objects that are highly sought after to develop biomaterials or tend to replicate the complex structure of native mineralized extracellular matrices.

Deposition of Intact and Active Proteins In Vacuo Using Large Argon Cluster Ion Beams.

Providing inert materials with a biochemical function, for example using proteins, is a cornerstone technology underlying many applications. However, the controlled construction of protein thin films remains a major challenge. Here, an innovative solvent-free approach for protein deposition is reported, using lysozyme as a model. By diverting a time-of-flight secondary ion mass spectrometer (ToF-SIMS) from its standard analytical function, large argon clusters were used to achieve protein transfer. A target consisting of a pool of proteins was bombarded with 10 keV Ar5000+ ions, and the ejected proteins were collected on a silicon wafer. The ellipsoidal deposition pattern was evidenced by ToF-SIMS analysis, while SDS-PAGE electrophoresis confirmed the presence of intact lysozyme on the collector. Finally, enzymatic activity assays demonstrated the preservation of the three-dimensional structure of the transferred proteins. These results pave the way to well-controlled protein deposition using ion beams and to the investigation of more complex multilayer architectures.

Hybrid chemoenzymatic heterogeneous catalyst prepared in one step from zeolite nanocrystals and enzyme-polyelectrolyte complexes.

The combination of inorganic heterogeneous catalysts and enzymes, in so-called hybrid chemoenzymatic heterogeneous catalysts (HCEHCs), is an attractive strategy to effectively run chemoenzymatic reactions. Yet, the preparation of such bifunctional materials remains challenging because both the inorganic and the biological moieties must be integrated in the same solid, while preserving their intrinsic activity. Combining an enzyme and a zeolite, for example, is complicated because the pores of the zeolite are too small to accommodate the enzyme and a covalent anchorage on the surface is often ineffective. Herein, we developed a new pathway to prepare a nanostructured hybrid catalyst built from glucose oxidase and TS-1 zeolite. Such hybrid material can catalyse the in situ biocatalytic formation of H2O2, which is subsequently used by the zeolite to trigger the epoxidation of allylic alcohol. Starting from an enzymatic solution and a suspension of zeolite nanocrystals, the hybrid catalyst is obtained in one step, using a continuous spray drying method. While enzymes are expectedly unable to resist the conditions used in spray drying (temperature, shear stress, etc.), we leverage on the preparation of "enzyme-polyelectrolyte complexes" (EPCs) to increase the enzyme stability. Interestingly, the use of EPCs also prevents enzyme leaching and appears to stabilize the enzyme against pH changes. We show that the one-pot preparation by spray drying gives access to hybrid chemoenzymatic heterogeneous catalysts with unprecedented performance in the targeted chemoenzymatic reaction. The bifunctional catalyst performs much better than the two catalysts operating as separate entities. We anticipate that this strategy could be used as an adaptable method to prepare other types of multifunctional materials starting from a library of functional nanobuilding blocks and biomolecules.

Correction: Hybrid chemoenzymatic heterogeneous catalyst prepared in one step from zeolite nanocrystals and enzyme-polyelectrolyte complexes.

[This corrects the article DOI: 10.1039/D0NA00834F.].

Large cluster ions: soft local probes and tools for organic and bio surfaces.

Ionised cluster beams have been produced and employed for thin film deposition and surface processing for half a century. In the last two decades, kiloelectronvolt cluster ions have also proved to be outstanding for surface characterisation by secondary ion mass spectrometry (SIMS), because their sputter and ion yields are enhanced in a non-linear fashion with respect to monoatomic projectiles, with a resulting step change of sensitivity for analysis and imaging. In particular, large gas cluster ion beams, or GCIB, have now become a reference in organic surface and thin film analysis using SIMS and X-ray photoelectron spectroscopy (XPS). The reason is that they induce soft molecular desorption and offer the opportunity to conduct damageless depth-profiling and 3D molecular imaging of the most sensitive organic electronics and biological samples, with a nanoscale depth resolution. In line with these recent developments, the present review focuses on rather weakly-bound, light-element cluster ions, such as noble or other gas clusters, and water or alcohol nanodroplets (excluding clusters made of metals, inorganic salts or ionic liquids) and their interaction with surfaces (essentially, but not exclusively, organic). The scope of this article encompasses three aspects. The first one is the fundamentals of large cluster impacts with surfaces, using the wealth of information provided by molecular dynamics simulations and experimental observations. The second focus is on recent applications of large cluster ion beams in surface characterisation, including mass spectrometric analysis and 2D localisation of large molecules, molecular depth-profiling and 3D molecular imaging. Finally, the perspective explores cutting edge developments, involving (i) new types of clusters with a chemistry designed to enhance performance for mass spectrometry imaging, (ii) the use of cluster fragment ion backscattering to locally retrieve physical surface properties and (iii) the fabrication of new biosurface and thin film architectures, where large cluster ion beams are used as tools to transfer biomolecules in vacuo from a target reservoir to any collector substrate.

Protein-based polyelectrolyte multilayers.

The immobilization of proteins to impart specific functions to surfaces is topical for chemical engineering, healthcare and diagnosis. Layer-by-Layer (LbL) self-assembly is one of the most used method to immobilize macromolecules on surfaces. It consists in the alternate adsorption of oppositely charged species, resulting in the formation of a multilayer. This method in principle allows any charged object to be immobilized on any surface, from aqueous solutions. However, when it comes to proteins, the promises of versatility, simplicity and universality that the LbL approach holds are unmet due to the heterogeneity of protein properties. In this review, the literature is analyzed to make a generic approach emerge, with a view to facilitate the LbL assembly of proteins with polyelectrolytes (PEs). In particular, this review aims at guiding the choice of the PE and the building conditions that lead to the successful growth of protein-based multilayered self-assemblies.

Self-Reorganizing Multilayer to Release Free Proteins from Self-Assemblies.

The deconstruction of self-assemblies based on proteins and polyelectrolytes (PEs) and the subsequent release of intact proteins require either a switch from attractive to repulsive mode or particular PE properties (degradability, responsiveness, or differential affinity). Here, an interfacial self-assembly made of three charged species, i.e., a strong polyacid complexed with a protein and a weak polybase, is shown to self-reorganize upon a shift in pH. When the pH takes a value that is one pH unit lower than the pKa of the weak polybase, the two PEs associate, thereby releasing the protein. The disassembly thus relies on associative forces rather than on the alteration of the protein-PE coupling strength. Hence, it allows the release of a protein using two simple PEs. The method is illustrated for lysozyme, which recovered up to half of its initial bioactivity after release. In contrast, a control self-assembled film that could not reorganize maintained only about 21% of the protein bioactivity after disassembly. This versatile approach is valuable for drug delivery devices and biomaterials as it allows the release of large numbers of active protein molecules.

Use of a quartz crystal microbalance platform to study protein adsorption on aluminum hydroxide vaccine adjuvants: Focus on phosphate-hydroxide ligand exchanges.

Aluminum hydroxide (AH) salts are widely used as vaccine adjuvants and controlling antigen-AH interactions is a key challenge in vaccine formulation. In a previous work, we have developed a quartz crystal microbalance (QCM) platform, based on stable AH-coated sensors, to explore the mechanisms of model antigen adsorption. The QCM study of bovine serum albumin (BSA) adsorption at different pH and ionic strength (I) values showed that protein adsorption on AH adjuvant at physiological pH cannot be explained mainly by electrostatic interactions, in contrast with previous reports. Here, we exploit further the developed QCM platform to investigate the role of phosphate-hydroxyl ligand exchanges in the adsorption mechanism of BSA, human serum albumin (HSA) and ovalbumin (OVA) on two commercial AH adjuvants. BSA adsorption decreased on immobilized AH particles previously treated with KH2PO4, highlighting the role of exchangeable sites on AH particles in the adsorption process. BSA and OVA were dephosphorylated by treatment with an acid phosphatase to decrease their phosphate content by about 80% and 25%, respectively. Compared to native BSA, adsorption of dephosphorylated BSA decreased significantly on one AH adjuvant at pH 7. Adsorption of dephosphorylated OVA was comparable to the one of native OVA. Further QCM assays showed that phospho-amino acids (PO4-serine and PO4-threonine) displaced previously adsorbed BSA and OVA from AH particles in conditions that were depending on the protein and the AH. Taken together, these observations suggest that phosphate-hydroxyl ligand exchange is an important adsorption mechanism of proteins on AH. These results moreover confirm that the developed AH-coated QCM sensors offer a new platform for the study of antigen adsorption, to the benefit of vaccine formulation.

Mixed Polymer Brushes for the Selective Capture and Release of Proteins.

Selective protein adsorption is a key challenge for the development of biosensors, separation technologies, and smart materials for medicine and biotechnologies. In this work, a strategy was developed for selective protein adsorption, based on the use of mixed polymer brushes composed of poly(ethylene oxide) (PEO), a protein-repellent polymer, and poly(acrylic acid) (PAA), a weak polyacid whose conformation changes according to the pH and ionic strength of the surrounding medium. A mixture of lysozyme (Lyz), human serum albumin (HSA), and human fibrinogen (Fb) was used to demonstrate the success of this strategy. Polymer brush formation and protein adsorption were monitored by quartz crystal microbalance, whereas protein identification after adsorption from the mixture was performed by time-of-flight secondary ion mass spectrometry (ToF-SIMS) with principal component analysis and gel electrophoresis with silver staining. For the ToF-SIMS measurements, adsorption was first performed from single-protein solutions in order to identify characteristic peaks of each protein. Next, adsorption was performed from the mixture of the three proteins. Proteins were also desorbed from the brushes and analyzed by gel electrophoresis with silver staining for further identification. Selective adsorption of Lyz from a mixture of Lyz/HSA/Fb was successfully achieved at pH 9.0 and ionic strength of 10-3 M, while Lyz and HSA, but not Fb, were adsorbed at ionic strength 10-2 M and pH 9.0. The results demonstrate that by controlling the ionic strength, selective adsorption can be achieved from protein mixtures on PEO/PAA mixed brushes, predominantly because of the resulting control on electrostatic interactions. In well-chosen conditions, the selectively adsorbed proteins can also be fully recovered from the brushes by a simple ionic strength stimulus. The developed systems will find applications as responsive biointerfaces in the fields of separation technologies, biosensing, drug delivery, and nanomedicine.

Antimicrobial peptide encapsulation and sustained release from polymer network particles prepared in supercritical carbon dioxide.

Antimicrobial peptide loaded poly(2-hydroxyethyl methacrylate) particles were synthesized in supercritical carbon dioxide via one-pot free-radical dispersion polymerisation of 2-hydroxyethyl methacrylate and a cross-linker. Discrete particles with a well-defined spherical morphology and a diameter as low as 450 nm have been obtained in mild conditions. The encapsulation and release of the peptide were confirmed by antimicrobial tests that demonstrated for the first time a sustained release of the peptide from poly(2-hydroxyethyl methacrylate) microgels prepared by one-pot dispersion polymerization in supercritical carbon dioxide and then dispersed in water.

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

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.

Sulindac encapsulation and release from functional poly(HEMA) microparticles prepared in supercritical carbon dioxide.

Sulindac loaded poly(HEMA) cross-linked microparticles were synthesized via one-pot free-radical dispersion polymerisation in supercritical carbon dioxide (scCO2) in presence of photocleavable diblock stabilisers based on polyethylene oxide (PEO) and poly(heptadecafluorodecyl acrylate) (PFDA) bearing a o-nitrobenzyl photosensitive junction (hv) (PEO-hv-PFDA), and ethylene glycol dimethacrylate (EGDMA) as cross-linker. Poly(HEMA) cross-linked microparticles either empty or sulindac loaded were obtained with well-defined spherical morphology with the sizes between 250 and 350 nm. Additionally, upon UV-photolysis the stabiliser on the surface was cleaved which permits to microparticles to be redispersed in water leading to water swollen microgels about 2.1-3.6 µm. Moreover, the release behaviour from obtained microgels indicated the sustained release of sulindac over 10 days. Besides, the surface modification after UV-photolysis was studied and proved that the particles can be functionalised with further chemistries.

Reversible Protein Adsorption on Mixed PEO/PAA Polymer Brushes: Role of Ionic Strength and PEO Content.

Proteins at interfaces are a key for many applications in the biomedical field, in biotechnologies, in biocatalysis, in food industry, etc. The development of surface layers that allow to control and manipulate proteins is thus highly desired. In previous works, we have shown that mixed polymer brushes combining the protein-repellent properties of poly(ethylene oxide) (PEO) and the stimuli-responsive adsorption behavior of poly(acrylic acid) (PAA) could be synthesized and used to achieve switchable protein adsorption. With the present work, we bring more insight into the rational design of such smart thin films by unravelling the role of PEO on the adsorption/desorption of proteins. The PEO content of the mixed PEO/PAA brushes was regulated, on the one hand, by using PEO with different molar masses and, on the other hand, by varying the ratio of PEO and PAA in the solutions used to synthesize the brushes. The influence of ionic strength on the protein adsorption behavior was also further examined. The behavior of three proteins-human serum albumin, lysozyme, and human fibrinogen, which have very different size, shape, and isoelectric point-was investigated. X-ray photoelectron spectroscopy, quartz crystal microbalance, atomic force microscopy, and streaming potential measurements were used to characterize the mixed polymer brushes and, in particular, to estimate the fraction of each polymer within the brushes. Protein adsorption and desorption conditions were selected based on previous studies. While brushes with a lower PEO content allowed the higher protein adsorption to occur, fully reversible adsorption could only be achieved when the PEO surface density was at least 25 PEO units per nm2. Taken together, the results increase the ability to finely tune protein adsorption, especially with temporal control. This opens up possibilities of applications in biosensor design, separation technologies, nanotransport, etc.