Analytical protocols for separation and electron microscopy of nanoparticles interacting with bacterial cells

An important step toward understanding interactions between nanoparticles (NPs) and bacteria is the ability to directly observe NPs interacting with bacterial cells. NPbacteria mixtures typical in nanomedicine, however, are not yet amendable for direct imaging in solution. Instead, evidence of NPcell interactions must be preserved in derivative (usually dried) samples to be subsequently revealed in high-resolution images, e.g., via scanning electron microscopy (SEM). Here, this concept is realized for a mixed suspension of model NPs and Staphylococcus aureus bacteria. First, protocols for analyzing the relative colloidal stabilities of NPs and bacteria are developed and validated based on systematic centrifugation and comparison of colony forming unit (CFU) counting and optical density (OD) measurements. Rate-dependence of centrifugation efficiency for each component suggests differential sedimentation at a specific predicted rate as an effective method for removing free NPs after co-incubation; the remaining fraction comprises bacteria with any associated NPs and can be examined, e.g., by SEM, for evidence of NPbacteria interactions. These analytical protocols, validated by systematic control experiments and high-resolution SEM imaging, should be generally applicable for investigating NPbacteria interactions.financial support from the following sources: grant SFRH/BPD/47693/2008 from the Portuguese Foundation for Science and Technology (FCT); FCT Strategic Project PEst-OE/EQB/LA0023/2013; project “BioHealth Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124-FEDER-000027, cofunded by the Programa Operacional Regional do Norte (ON.2−O Novo Norte), QREN, FEDER; project “Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB”, ref. FCOMP-01-0124-FEDER- 027462

Hydrocalumite Thin Films for Polyphenol Biosensor Elaboration

—Hybrid thin films based on Hydrocalu-mite (Ca 2 AlCl layered double hydroxide LDH) and tyrosinaseenzyme have been used for the elaboration of a high sensitive amperometric biosensor detecting polyphenols extracted from green tea. Structural properties of LDH nanomaterials were characterized by X-ray powder diffraction and Infra-Red spectroscopy, confirming its crystalline phase and chemical composition. Ca 2 AlCl-LDHs-thin films were deposited by spin-coating, and studied by atomic force microscopy to obtain information about the surface morphology of this host matrix before and after enzyme's immobilization. Electrochemical study using cyclic voltammetry and chronoamperometry shows good performances of the built-in biosensor with a high sensitivity for polyphenols concentrations ranging from 24 pM to 2.4 µM and a limit of detection of 1.2 pM. Index Terms—Amperometric biosensor, layered double hydroxide, tyrosinase, hybrid nanomaterial, atomic force microscopy

Isolation and molecular identification of Vibrio spp. by sequencing of 16S rDNA from seafood, meat and meat products in Libya

The genus Vibrio includes several food-borne pathogens that cause a spectrum of clinical conditions including septicemia, cholera and milder forms of gastroenteritis. Several Vibrio spp. are commonly associated with food-borne transmission including Vibrio cholerae, Vibrio parahemolyticus, and Vibrio vulnificus. Microbiological analysis for enumeration and isolation of Vibrio spp. were carried out for a total of 93 samples of seafood, meat and meat products from different geographic localities in Libya (Tripoli, Regdalin, Janzour and Tobruk). Vibrio spp. were detected by conventional cultural and molecular method using PCR and sequencing of 16S rDNA. Out of the 93 cultured samples only 48 (51.6%) yielded colonies on Thiosulfate Citrate Bile Salt agar (TCBS) with culture characteristics of Vibrio spp. More than half (n=27) of processed seafood samples (n=46) yielded colonies on TCBS, while only 44.6% of samples of meat and meat products showed colonies on TCBS. Among cultured seafood samples, the highest bacterial count was recorded in clam with a count of 3.8 х104 CFU\g. Chicken burger samples showed the highest bacterial count with 6.5 х104 CFU\g. Molecular analysis of the isolates obtained in this study, showed that 11 samples out of 48 (22.9%) were Vibrio spp. Vibrio parahemolyticus was isolated from camel meat for the first time. This study is an initial step to provide a baseline for future molecular research targeting Vibrio spp. foodborne illnesses. This data will be used to provide information on the magnitude of such pathogens in Libyan seafood, meat and meat products.Keywords: 16S rDNA, Libya, Meat, Seafood, Vibri

Amperometric Polyphenol Biosensor Based on Tyrosinase Immobilization on CoAl Layered Double Hydroxide Thins Films

International audienceAn amperometric biosensor based on tyrosinase immobilized on the sensor surface has been used for the detection of polyphenols extracted from green tea. The immobilization was ensured by the crosslinking method on thins films of CoAlSO 4 layered double hydroxide recovering screen-printed gold electrodes. Electrochemical measurements show that this biosensor is able to detect tea polyphenols by following the reduction of compounds enzymatically generated. Its response is linear in the concentration range of [0-2,4 µM] with high sensitivity and stability, since it retains 90% of its original response after 20 days

Membrane damages in bacteria interacting with silica nanoparticles revealed by AFM

International audienceNanoparticles (NPs) can interact with biological systems, with either negative or positive consequences (potential risks or elimination of pathogenic bacteria). In this context, we investigate the morphology and physico-chemical properties of Escherichia coli bacteria interacting with silica NPs by Atomic Force Microscopy (AFM), this method providing access to topographic information and local rheological properties at the nm scale (with a discrimination between “hard”, NPs, and “soft”, bacteria, materials), either in air or physiological environment. AFM images show that silica NPs tend to aggregate around bacteria, their further action depending on their diameter. The presence of big NPs (100 and 200 nm) does not change E. coli morphology, bacteria remaining rod-shaped and high. The bacterial external membrane keeps also its organization in domains, suggesting that such NPs are too voluminous to penetrate into bacteria. On the contrary, in the presence of small NPs (4 and 10 nm) bacteria adopt unusual spherical shapes, some of them even suffering from a partial collapse, leading to the release of cellular compounds. The external membrane is also disturbed, exhibiting spherical aggregates, which could be due to a reorganization of lipopolysaccharides present in this membrane

Membrane damages in bacteria interacting with silica nanoparticles revealed by AFM

Nanoparticles (NPs) can interact with biological systems, with either negative or positive consequences (potential risks or elimination of pathogenic bacteria). In this context, we investigate the morphology and physico-chemical properties of Escherichia coli bacteria interacting with silica NPs by Atomic Force Microscopy (AFM), this method providing access to topographic information and local rheological properties at the nm scale (with a discrimination between “hard”, NPs, and “soft”, bacteria, materials), either in air or physiological environment. AFM images show that silica NPs tend to aggregate around bacteria, their further action depending on their diameter. The presence of big NPs (100 and 200 nm) does not change E. coli morphology, bacteria remaining rod-shaped and high. The bacterial external membrane keeps also its organization in domains, suggesting that such NPs are too voluminous to penetrate into bacteria. On the contrary, in the presence of small NPs (4 and 10 nm) bacteria adopt unusual spherical shapes, some of them even suffering from a partial collapse, leading to the release of cellular compounds. The external membrane is also disturbed, exhibiting spherical aggregates, which could be due to a reorganization of lipopolysaccharides present in this membrane

Innovative Spirulina Platensis-Based Love Wave Microsensor for the Detection of Heavy Metal in Liquid Medium

A dried biomass of Arthrospira platensis, called Spirulina (Sp), a filamentous cyanobacterium, a blue-green microalga, is known to bind a wide range of heavy metals [1]. In this work, we propose an innovative approach, based on Spirulina as bioreceptor, combined with highly sensitive Love wave platform for the real-time detection of HM in liquid medium. The real time response to various concentrations of Cadmium (Cd2+) and Mercury (Hg2+), pumped through the microchannels, has been investigated. A detection limit as low as 10-12 mol.l-1 has been obtained. This response has been attributed to changes of microalgae visco-elastic equivalent parameters in presence of heavy metals

Innovative Spirulina Platensis-Based Love Wave Microsensor for the Detection of Heavy Metal in Liquid Medium

A dried biomass of Arthrospira platensis, called Spirulina (Sp), a filamentous cyanobacterium, a blue-green microalga, is known to bind a wide range of heavy metals [1]. In this work, we propose an innovative approach, based on Spirulina as bioreceptor, combined with highly sensitive Love wave platform for the real-time detection of HM in liquid medium. The real time response to various concentrations of Cadmium (Cd2+) and Mercury (Hg2+), pumped through the microchannels, has been investigated. A detection limit as low as 10-12 mol.l-1 has been obtained. This response has been attributed to changes of microalgae visco-elastic equivalent parameters in presence of heavy metals