Claire-Marie Pradier

Antibacterial surfaces developed from bio-inspired approaches.

Prevention of bacterial adhesion and biofilm formation on the surfaces of materials is a topic of major medical and societal importance. Various synthetic approaches based on immobilization or release of bactericidal substances such as metal derivatives, polyammonium salts and antibiotics were extensively explored to produce antibacterial coatings. Although providing encouraging results, these approaches suffer from the use of active agents which may be associated with side-effects such as cytotoxicity, hypersensibility, inflammatory responses or the progressive alarming phenomenon of antibiotic resistance. In addition to these synthetic approaches, living organisms, e.g. animals and plants, have developed fascinating strategies over millions of years to prevent efficiently the colonization of their surfaces by pathogens. These strategies have been recently mimicked to create a new generation of bio-inspired biofilm-resistant surfaces. In this review, we discuss some of these bio-inspired methods devoted to the development of antibiofilm surfaces. We describe the elaboration of antibacterial coatings based on natural bactericidal substances produced by living organisms such as antimicrobial peptides, bacteriolytic enzymes and essential oils. We discuss also the development of layers mimicking algae surfaces and based on anti-quorum-sensing molecules which affect cell-to-cell communication. Finally, we report on very recent strategies directly inspired from marine animal life and based on surface microstructuring.

Peptide Interactions with Metal and Oxide Surfaces

Increasing interest in bio-interfaces for medical, diagnostic, or biotechnology applications has highlighted the critical scientific challenge behind both the understanding and control of protein-solid surface interactions. In this context, this Account focuses on the molecular-level characterization of the interactions of peptides, ranging in size from a few amino acids to long sequences, with metal and oxide surfaces. In this Account, we attempt to fill the gap between the well-known basic studies of the interaction of a single amino acid with well-defined metal surfaces and the investigations aimed at controlling biocompatibility or biofilm growth processes. We gather studies performed with surface science tools and macroscopic characterization techniques along with those that use modeling methods, and note the trends that emerge. Sulfur drives the interaction of cysteine-containing peptides with metal surfaces, particularly gold. Moreover, intermolecular interactions, such as hydrogen bonds may induce surface self assembly and chiral arrangements of the peptide layer. Depending on the solvent pH and composition, carboxylates or amino groups may also interact with the surface, which could involve conformational changes in the adsorbed peptide. On oxide surfaces such as titania or silica, researchers have identified carboxylate groups as the preferential peptide binding groups because of their strong electrostatic interactions with the charged surface. In high molecular weight peptides, systematic studies of their interaction with various oxide surfaces point to the preferential interaction of certain peptide sequences: basic residues such as arginine assume a special role. Researchers have successfully used these observations to synthesize adhesive sequences and initiate biomineralization. Studies of the interaction of peptides with nanoparticles have revealed similar binding trends. Sulfur-containing peptides adhere preferentially to gold nanoparticles. Peptides containing aromatic nitrogen also display a high affinity for various inorganic nanoparticles. Finally, we describe a novel class of peptides, genetically engineered peptides for inorganics (GEPIs), which are selected from a phage display protocol for their high binding affinity for inorganic surfaces. Extended investigations have focused on the mechanisms of the molecular binding of these peptides to solid surfaces, in particular the high binding affinity of some sulfur-free sequences of GEPIs to gold or platinum surfaces. We expect that this clearer view of the possible preferential interactions between peptides and inorganic surfaces will facilitate the development of new, more focused research in various fields of biotechnology, such as biocompatibility, biomimetics, or tissue engineering.

The antibacterial activity of Magainin I immobilized onto mixed thiols Self-Assembled Monolayers

An antibacterial peptide, Magainin I, was covalently bound to a mixed 11-mercaptoundecanoïc acid (MUA) and 6-mercaptohexanol (C6OH) (ratio 1:3) Self-Assembled Monolayer (SAM) on gold surfaces. Each step of the surface functionalization was characterized by Polarization Modulation Reflection Absorption InfraRed Spectroscopy (PM-RAIRS) and X-ray Photoelectron Spectroscopy (XPS). The antibacterial activity of the anchored Magainin was tested against three Gram-positive bacteria (Listeria ivanovii, Enterococcus faecalis and Staphylococcus aureus), and the results revealed that the adsorbed Magainin I reduced by more than 50% the adhesion of bacteria at the surface, together with the killing of the bacteria that nonetheless adhered to the surface. No release of the peptide was observed upon contact with the bacterial suspension; the activity has proven to be persistent overtime, up to six months after the first use.

Kinetics of conditioning layer formation on stainless steel immersed in seawater

Adhesion of microorganisms to surfaces in marine environments leads to biofouling. The deleterious effects of biofilm growth in the marine environment are numerous and include energy losses due to increased fluid frictional resistance or to increased heat transfer resistance, the risk of corrosion induced by microorganisms, loss of optical properties, and quality control and safety problems. Antifouling agents are generally used to protect surfaces from such a biofilm. These agents are toxic and can be persistent, causing harmful environmental and ecological effects. Moreover, the use of biocides and regular cleaning considerably increase the maintenance costs of marine industries. An improved knowledge of bio‐film adhesion mechanisms is needed for the development of an alternative approach to the currently used antifouling agents. The aim of this study is to characterise the chemical composition of the molecules first interacting with stainless steel during the period immediately following immersion in natural seawater and to elucidate the kinetics of the adsorbtion process. Proteins are shown to adhere very rapidly, closely followed by carbohydrates. The distribution on the surface of organic molecules is also examined. The ad‐sorbate on the surface is not a continuous film but a heterogeneous deposit, whose average thickness varies widely. The cleaning procedures used affect the adsorption kinetics. In particular, cleaning with hexane results in slower adsorption of nitrogen‐containing species than does cleaning in acetone.

Surface Plasmon Resonance Biosensors Incorporating Gold Nanoparticles

SPR biosensing is increasingly popular for the detection of a multitude of biomolecules. It offers label-free detection and study of proteins, nucleic acids, and other biomolecules in real time. A recent trend involves incorporation of AuNPs, either within the sensing surface itself or as signal enhancing tagging molecules. The importance of AuNP and detecting agent spacing is described and techniques using macromolecular spacing aids are highlighted. Recent methods to enhance SPR detection capabilities using gold nanoparticles are reviewed, as well as device fabrication and the results of incorporation. SPR detection is a highly versatile method for the detection of biomolecules and, with the incorporation of AuNPs, shows promise in extending it to a number of new applications.

Grafting of Lysozyme and/or Poly(ethylene glycol) to Prevent Biofilm Growth on Stainless Steel Surfaces

In the aim of protecting stainless steel surfaces against protein and/or bacterial adhesion, thin films including the glycosidase hen egg white lysozyme (HEWL) and/or the synthetic polymer poly(ethylene glycol) (PEG) were covalently coated onto flat substrates by wet chemical processes. Chemical grafting of both species was carried out by covalent binding to surfaces pretreated by the polyamine poly(ethylene imine) (PEI). Surfaces were characterized at each step of functionalization by means of reflection-absorption infrared spectroscopy by modulation of polarization (PM-RAIRS) and X-ray photoelectron spectroscopy (XPS) to determine the atomic and molecular composition of the interfaces, respectively. Then, the ability of the so-modified surfaces to prevent protein adsorption and bacterial adhesion together with their biocide properties were demonstrated by three local tests employing bovine serum albumin (BSA), and the bacteria Listeria ivanovii and Micrococcus luteus. A new test was implemented to assess the local enzymatic properties of HEWL. Cografting of PEG and HEWL resulted in a surface with both antiadhesion and antibacterial properties.

Optimized grafting of antimicrobial peptides on stainless steel surface and biofilm resistance tests

Antibacterial peptides, magainin I and nisin were covalently bound to stainless steel surfaces. Several procedures of surface functionalisation processes have been investigated and optimized, each step being characterized by polarization modulation reflection absorption infrared spectroscopy (PM-RAIRS) and X-ray photoemission spectroscopy (XPS). Grafting of antibacterial peptides was successfully achieved by a 3 steps functionalisation process on a chitosan polymeric layer. The antibacterial activity of the anchored magainin and nisin was tested against a gram-positive bacteria, Listeria ivanovii, i.e., the possible survival and attachment of this bacteria, was characterized on modified stainless steel surfaces. The results revealed that the adsorbed peptides reduced the adhesion of bacteria on the functionalised stainless steel surface.

In-depth investigation of protein adsorption on gold surfaces: Correlating the structure and density to the efficiency of the sensing layer

Protein A (PrA), mouse monoclonal anti-IgG antibody (SAb) and deglycosylated avidin (NAV) were adsorbed on gold surfaces to capture the model rabbit IgG and build three immunosensing platforms. The assembling of immunosensors, their specificity, and the receptor accessibility were monitored by polarization modulation reflection-absorption infrared spectroscopy (PM-RAIRS) and quartz crystal microbalance with dissipation measurement (QCM-D) at each step. Combining these two techniques allows us to compare both chemical and structural properties of the sensing layers with the former bringing chemical and semiquantitative information on the grafted protein layers, whereas the latter, in addition to the mass uptake, enables us to take the layer rigidity into account. Grafting of the three capture proteins to the transducer surfaces, covered with appropriate self-assembled monolayers, yielded protein layers with variable properties. NAV formed a dense and rigid molecular layer, likely containing protein aggregates, whereas the amount of PrA was below one monolayer resulting in a flexible layer. The amount of immobilized rabbit IgG was different for the three systems with the densest capture protein layer exhibiting the lowest binding capacity. The accessibility of antibodies on the resulting immunosensors measured by interaction with a secondary antirabbit IgG antibody was found to be closely dependent on their coverage as well as on the rigidity of the protein layer. The overall study provides in-depth information on three of the most common immunosensor recognition interfaces and demonstrates the crucial influence of both structure and density of the protein layer on the efficiency of the molecular recognition phenomena.

Specific binding of avidin to biotin immobilised on modified gold surfaces: Fourier transform infrared reflection absorption spectroscopy analysis

Abstract The immobilisation of biomolecules on gold surfaces has attracted a lot of attention due to the various ways of transducing biorecognition events at such surfaces and of course to its broad field of applications. The current challenge is two-fold: first, to produce a functionalised surface able to bind a given biomolecule in a complex environment, but also resistant to nonspecific adsorption; second, to assess the protein–ligand interaction by a suitable physical method. In our attempt to conceive a new type of biosensor, the biotin/avidin system was chosen as a model of biological (ligand/receptor) interaction. The first task was achieved by developing a two-step procedure, briefly consisting in the chemisorption of the short diamine disulfide cystamine to a gold substrate followed by chemical coupling with the N -succinimidyl ester of biotin. The presence of biotin molecules both specifically and unspecifically bound to the gold surface was assessed by Fourier transform infrared reflection absorption spectroscopy (FT-IRRAS) and XPS. Undesired nonspecific binding of biotin was minimised by increasing the chain length of the chemisorbed amine to which was coupled the N -succinimidyl ester of biotin. Chemical characterisation of the adsorbed layer was performed after each step by FT-IRRAS. For the second task, the protein avidin was labelled with an alkyne Co 2 (CO) 6 probe, a transition metal carbonyl entity that yields three characteristic ν CO signals far from the peptide region of absorption, before its interaction with the modified surface. Molecular recognition was checked and quantified by FT-IRRAS. The occurrence of nonspecific adsorption of avidin was measured by exposing the biotinylated substrates to a solution of labelled avidin pre-saturated by biotin. Methods to reduce nonspecific binding of avidin were also shown.

Piezoelectric immunosensor for direct and rapid detection of staphylococcal enterotoxin A (SEA) at the ng level

A direct, label-free immunosensor was designed for the rapid detection and quantification of staphylococcal enterotoxin A (SEA) in buffered solutions using quartz crystal microbalance with dissipation (QCM-D) as transduction method. The sensing layer including the anti-SEA antibody was constructed by chemisorption of a self-assembled monolayer of cysteamine on the gold electrodes placed over the quartz crystal sensor followed by activation of the surface amino groups with the rigid homobifunctional cross-linker 1,4-phenylene diisothiocyanate (PDITC) and covalent linking of binding protein (protein A or protein G). Four anti-SEA antibodies (two of which from commercial source) have been selected to set up the most sensitive detection device. With the optimized sensing layer, a standard curve for the direct assay of SEA was established from QCM-D responses within a working range of 50-1000 or 2000 ngml(-1) with a detection limit of 20 ngml(-1). The total time for analysis was 15 min. Using a sandwich type assay, the response was ca. twice higher and consequently the lowest measurable concentration dropped down to 7 ngml(-1) for a total assay time of 25 min.