Label-free biosensors are ideally suited for the quantitative analysis of specific interactions among biomolecules or of biomolecules with drugs, as well as for quantitation of diagnostic markers in biofluids. In contrast to the label-dependent methods, a new assay for a particular prey molecule can be set up within few minutes by immobilizing the corresponding bait molecule on the sensor surface, using one of the common immobilization procedures. Unfortunately, the extensive application of label-free biosensors is still hampered by the fact that the immobilization of the bait molecule is usually irreversible; for that reason, a new chip (which is expensive) is required for every successful or futile attempt. Here, we present a general method for the switchable immobilization of biotinylated bait molecules on a new desthiobiotin surface, using wild-type streptavidin as a robust bridge between the chip and the biotinylated bait. The immobilization of the bait is very stable, so that many cycles of prey injection and subsequent prey removal can be carried out. For the latter, common reagents like HCl, NaCO, glycine buffer, or SDS are employed. When desired, however, streptavidin plus the biotinylated bait can be completely removed by 3min injections of biotin, guanidinium thiocyanate, pepsin, and SDS, which makes it possible to immobilize new biotinylated bait. The number of in situ regeneration cycles is unlimited during the lifetime of the chip (2-3 weeks). One chip can easily be shared by many users with unrelated tasks (as is typical in academics), or used for the fully automated screening of many different interactions (for example in pharmaceutical research). In comparison to other regenerative chips, the new chip surface has much wider applicability and all of its structural and functional parameters have been disclosed.
The aim of this study was to investigate the effects of garlic oil, cinnamaldehyde, carvacrol, thymol, and thyme oil on growth and biofilm formation of Escherichia coli and Salmonella serotypes, including field isolates from livestock animals. Minimum inhibitory concentrations (MIC) were determined using broth micro-dilution method. Biofilm biomass was assessed by measuring the attached biomass with microtiter plate assay and crystal violet (CV) staining. The strongest antimicrobial effects on E. coli serotypes were observed for thymol at 150 ppm, followed by carvacrol and cinnamaldehyde at 300 ppm and thyme oil at 600 ppm. Similar results were obtained with Salmonella serotypes except for carvacrol (MIC value at 150 ppm). Garlic oil showed no growth inhibition on serotypes of E. coli and Salmonella up to 10000 ppm. Cinnamaldehyde proved to be the most effective substance in reducing E. coli CV-biofilm formation at sub-MIC level with a threshold concentration of 5 ppm, followed by carvacrol, thymol, and thyme oil at 40 ppm and garlic oil at 10000 ppm. CV-biofilm formation of Salmonella serotypes at sub-MIC level was clearly reduced with 40 ppm cinnamaldehyde and 80 ppm carvacrol, thymol, and thyme oil. No reduction of CV-biofilm formation was observed with garlic oil. The present study demonstrates a strong antibacterial activity of cinnamaldehyde, carvacrol, thymol, and thyme oil. Similar response of field isolates and type strains to these phytogenics suggests a general effect within the bacterial species tested. All four substances were also able to reduce CV-biofilm formation at sub-MIC level. Investigating phytogenics with bacterial field isolates contributes to the development of feed additives as alternatives to antibiotics in animal feed to increase productivity and animal welfare in modern livestock production.
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