Legionella pneumophila sg1-sensing signal enhancement using a novel electrochemical immunosensor in dynamic detection mode.

This work presents a comparison between static and dynamic modes of biosensing using a novel microfluidic assay for continuous and quantitative detection of Legionella pneumophila sg1 in artificial water samples. A self-assembled monolayer of 16-amino-1-hexadecanethiol (16-AHT) was covalently linked to a gold substrate, and the resulting modified surface was used to immobilize an anti-Legionella pneumophila monoclonal antibody (mAb). The modified surfaces formed during the biosensor functionalization steps were characterized using electrochemical measurements and microscopic imaging techniques. Under static conditions, the biosensor exhibited a wide linear response range from 10 to 108 CFU/mL and a detection limit of 10 CFU/mL. Using a microfluidic system, the biosensor responses exhibited a linear relationship for low bacterial concentrations ranging from 10 to 103 CFU/mL under dynamic conditions and an enhancement of sensing signals by a factor of 4.5 compared to the sensing signals obtained under static conditions with the same biosensor for the detection of Legionella cells in artificially contaminated samples.

Ultrasensitive Electrochemical Sensors for PSA Detection: Related Surface Functionalization Strategies.

Prostate cancer is the most common male cancer in the world. The diagnosis, staging, prognosis and monitoring are usually done with Prostate Specific Antigen (PSA). Biosensors are emerging as a novel analytical technology for PSA detection. They provide several advantages for clinical applications and will benefit clinicians, patients and forensic workers in the future. Among them, electrochemical immunosensors have gained growing interests. Hence, their sensitivity is often improved by modifying them with nanoparticles especially iron oxide (IONP). Functionalized IONP attracted much attention in the fabrication of biosensing systems, due to their multiple properties, such as biocompatibility and signal amplification, and their ability to bind covalently to antibodies via their functional groups. In the present study, two electrochemical immunosensors were investigated for PSA detection. The first one was functionalized with 3- glycidoxypropyltrimethoxysilane self-assembled monolayer, while the second one was based on iron oxide nanoparticles functionalized with 3-aminopropyltriethoxysilane. Square wave voltammetry (SWV) has been investigated to follow-up the PSA detection in a phosphate buffer solution, in an artificial serum and in a human serum. The limit of detection (LOD) of both immunosensors was found of order of 10 fg/ml. When estimated in human serum this value increases up to 50 pg/ml.

AFM, CLSM and EIS characterization of the immobilization of antibodies on indium-tin oxide electrode and their capture of Legionella pneumophila.

The microscopic surface molecular structures and properties of monoclonal anti-Legionella pneumophila antibodies on an indium-tin oxide (ITO) electrode surface were studied to elaborate an electrochemical immunosensor for Legionella pneumophila detection. A monoclonal anti-Legionella pneumophila antibody (MAb) has been immobilized onto an ITO electrode via covalent chemical bonds between antibodies amino-group and the ring of (3-Glycidoxypropyl) trimethoxysilane (GPTMS). The functionalization of the immunosensor was characterized by atomic force microscopy (AFM), water contact angle measurement, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6](3-/4-) as a redox probe. Specific binding of Legionella pneumophila sgp 1 cells onto the antibody-modified ITO electrode was shown by confocal laser scanning microscopy (CLSM) imaging and EIS. AFM images evidenced the dense and relatively homogeneous morphology on the ITO surface. The formation of the complex epoxysilane-antibodies acting as barriers for the electron transfer between the electrode surface and the redox species in the solution induced a significant increase in the charge transfer resistance (Rct) compared to all the electric elements. A linear relationship between the change in charge transfer resistance (ΔRct=Rct after immunoreactions - Rct control) and the logarithmic concentration value of L. pneumophila was observed in the range of 5 × 10(1)-5 × 10(4) CFU mL(-1) with a limit of detection 5 × 10(1)CFU mL(-1). The present study has demonstrated the successful deposition of an anti-L. pneumophila antibodies on an indium-tin oxide surface, opening its subsequent use as immuno-captor for the specific detection of L. pneumophila in environmental samples.

Surface assembly on biofunctional magnetic nanobeads for the study of protein-ligand interactions.

One of the major challenges of proteomics today is to increase the power potential for the identification of as many proteins as possible and to characterize their interactions with specific free ligands (interactomics) or present on cell walls (cell marker), in order to obtain a global, integrated view of disease processes, cellular processes and networks at the protein level. The work presented here proposes the development of biofunctionalized magnetic nanobeads that might be used for interactomic investigations. The strategy consisted in immobilizing proteins via a non covalent technique that provides greater possibilities for the advent of faster, cheaper and highly miniaturizable protein analysis systems, in particular in situations where the amount of isolated protein is scarce (trace proteins). The advantage of the immobilization technique proposed here over more conventional covalent binding techniques is that it is versatile and universal (not protein specific) thus applicable to a wide range of proteins, in "mild" conditions that are non deleterious to the native structure and bioactivity of the immobilized protein. The feasibility of the technique was investigated using a model protein (streptavidin). The nanobeads were analyzed in size by light diffusion and transmission electronic spectroscopy, and in quantity of immobilized protein using a bioassay developed in the laboratory. Results are promising in that nanobeads exhibited good colloidal stability and surface concentrations in the monolayer range.