Electrospray deposition in vacuum as method to create functionally active protein immobilization on polymeric substrates

We demonstrate in this work the deposition of a large biological molecule (fibronectin) on polymeric substrates in a high vacuum environment using an electrospray deposition system. Fibronectin was deposited and its distribution and structure investigated and retention of function (ability to promote cell adhesion) on return to liquid environment is shown. AFM was used to monitor changes in the morphology of the surface before and after fibronectin deposition, whilst the biological activity of the deposited protein is assessed through a quantitative analysis of the biomolecular adhesion and migration of fibroblast cells to the modified surfaces. For the first time we have demonstrated that using high vacuum electrospray deposition it is possible to deposit large protein molecules on polymeric surfaces whilst maintaining the protein activity. The deposition of biological molecules such as proteins with the retention of their activity onto clean well-controlled surfaces under vacuum condition, offers the possibility for future studies utilizing high resolution vacuum based techniques at the atomic and molecular scale providing a greater understanding of protein–surface interface behaviour of relevance to a wide range of applications such as in sensors, diagnostics and tissue engineering

Bioheterojunction Effect on Fluorescence Origin and Efficiency Improvement of Firefly Chromophores

We propose the heterojunction effect in the analysis of the fluorescence mechanism of the firefly chromophore. Following this analysis, and with respect to the HOMO-LUMO gap alignment between the chromophore's functional fragments, three main heterojunction types (I, II, and I*) are identified. Time-dependent density-functional theory optical absorption calculations for the firefly chromophore show that the strongest excitation appears in the deprotonated anion state of the keto form. This can be explained by its high HOMO-LUMO overlap due to strong bio-heterojunction confinement. It is also found that the nitrogen atom in the thiazolyl rings, due to its larger electronegativity, plays a key role in the emission process, its importance growing when HOMO and LUMO overlap at its location. This principle is applied to enhance the chromophore's fluorescence efficiency and to guide the functionalization of molecular optoelectronic devices.Comment: 7 pages, 6 figure

GENERAL CHARACTERISTICS OF BIOLUMI- NESCENCE ASSAY OF INTRACELLULAR ATP Bioluminescence Assay for Cell Viability

ATP as an indicator of cell viability. Adenosine triphosphate (systematic name 9-β-D-ribofuranosyl adenine-5′-triphosphate or 9-β-D-ribofuranosyl-6-aminopurine-5′-triphosphate) − a nucleotide, an adenosine triphosphate ester that is a derivative of adenine and ribose (ATP) − is the main energy carrier in cells of all living organisms (mammals, microorganisms, plants, etc.) [1]. Cleavage of one or two phosphate groups that occurs during ATP hydrolysis is accompanied by the release of energy. In cells, ATP transfers energy to other molecules upon hydrolysis to its low-energy analogs (ADP and/or AMP), which, in turn, acquire energy by adding phosphate groups and transforming into ATP. Intracellular ATP content is the main indicator of cell viability. Upon cell death, ATP synthesis is the first to be arrested, while its hydrolysis can continue for some time, hence, the intracellular ATP content drops sharply to zero value. The ATP content in viable cells of microorganisms is quite high -it ranges from 500 to 10,000 µg per g of dry biomass Determination of ATP concentration using bioluminescence assay. There are various methods to determine ATP concentration: enzymatic methods with spectrophotometric detection, radioactive and chromatographic methods, and others. The bioluminescence ATP assay is the most sensitive, rapid, and selective. As far back as in the 1940s, it was shown that ATP is a required component in the reaction catalyzed by the firefly luciferase enzym

Assessment of Photodynamic Destruction of Escherichia coli O157:H7 and Listeria monocytogenes by Using ATP Bioluminescence

Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.