Development of the Pseudomonas syringae pv. morsprunorum Biofilm Monitored in Real Time Using Attenuated Total Reflection Fourier Transform Infrared Measurements in a Flow Cell Chamber.

Biofilms of sessile Pseudomonas syringae cells formed on top of plant host's leaves or fruits allow surviving harsh environmental conditions (desiccation) and improve their resistance to antibacterial treatments of crops. A better understanding of these biofilms can help minimize their effect on harvests. In the present study, infrared attenuated total reflection spectroscopy coupled with optical and confocal laser scanning microscopy has been applied for the first time to analyze Pseudomonas syringae pathovar morsprunorum biofilm development in real time. The biofilm development was observed within a spectral window 4000-800 cm-1 under constant flow conditions for 72 h. The kinetics of representative integrated band areas (nucleic acids with polysaccharides at 1141-1006 cm-1, amino acid side chains with free fatty acids at 1420-1380 cm-1, proteins at 1580-1490 cm-1, and lipids with proteins at 2935-2915 cm-1) were analyzed with regard to the observed biofilm structure and the following P. syringae biofilm developmental stages were attributed: The inoculation phase, washing of weakly attached bacteria closely followed by recolonization of the vacated surface, the restructuration phase, and finally the maturation phase.

Impact of electrolyte solution on electrochemical oxidation treatment of Escherichia coli K-12 by boron-doped diamond electrodes.

We studied the disinfection efficacy of boron-doped electrodes on Escherichia coli-contaminated water-based solutions in three different electrolytes, physiological solution (NaCl), phosphate buffer (PB), and phosphate buffer saline (PBS). The effect of the electrochemical oxidation treatment on the bacteria viability was studied by drop and spread plate cultivation methods, and supported by optical density measurements. We have found that bacterial suspensions in NaCl and PBS underwent a total inactivation of all viable bacteria within 10 min of the electrochemical treatment. By contrast, experiments performed in the PB showed a relatively minor decrease of viability by two orders of magnitude after 2 h of the treatment, which is almost comparable with the untreated control. The enhanced bacterial inactivation was assigned to reactive chlorine species, capable of penetrating the bacterial cytoplasmic membrane and killing bacteria from within.

Nanodiamond surface chemistry controls assembly of polypyrrole and generation of photovoltage.

Nanoscale composite of detonation nanodiamond (DND) and polypyrrole (PPy) as a representative of organic light-harvesting polymers is explored for energy generation, using nanodiamond as an inorganic electron acceptor. We present a technology for the composite layer-by-layer synthesis that is suitable for solar cell fabrication. The formation, pronounced material interaction, and photovoltaic properties of DND-PPy composites are characterized down to nanoscale by atomic force microscopy, infrared spectroscopy, Kelvin probe, and electronic transport measurements. The data show that DNDs with different surface terminations (hydrogenated, oxidized, poly-functional) assemble PPy oligomers in different ways. This leads to composites with different optoelectronic properties. Tight material interaction results in significantly enhanced photovoltage and broadband (1-3.5 eV) optical absorption in DND/PPy composites compared to pristine materials. Combination of both oxygen and hydrogen functional groups on the nanodiamond surface appears to be the most favorable for the optoelectronic effects. Theoretical DFT calculations corroborate the experimental data. Test solar cells demonstrate the functionality of the concept.

Determination of tumour biomarkers homovanillic and vanillylmandelic acid using flow injection analysis with amperometric detection at a boron doped diamond electrode.

A new method for the simultaneous determination of two tumour biomarkers, homovanillic (HVA) and vanillylmandelic acid (VMA), using flow injection analysis (FIA) with amperometric detection (AD) at a commercially available boron doped diamond electrode (BDDE) was developed. It was found that this method is suitable for the determination of HVA (in the presence of VMA) and VMA (in the presence of HVA) in optimum medium of Britton-Robinson buffer (0.04 mol L-1, pH 3.0). Calibration dependences consist of two linear parts for both biomarkers, the first one being in the concentration range from 1 to 10 µmol L-1 and the second one from 10 to 100 µmol L-1 (with obtained LODs 0.44 µmol L-1 for HVA and 0.34 µmol L-1 for VMA, respectively). To minimize any negative effects related to the passivation of the working electrode, suitable cleaning pulses (+2.4 V for 30 s) were imposed on the working electrode after each measurement. An attempt to use FIA with multiple pulse amperometric detection to determine both analytes in one run was not successful. Changing potentials in short intervals in multiple pulse detection probably results in mutual interaction of analytes and/or products of their electrochemical oxidation, thus preventing the application of this approach.

Cyclic Changes in the Amide Bands Within Escherichia coli Biofilms Monitored Using Real-Time Infrared Attenuated Total Reflection Spectroscopy (IR-ATR).

Contrary to the planktonic state of bacteria, their biofilm form represents severe complications in areas such as human medicine or food industry due to the increasing resistance against harsh conditions and treatment. In the present study, infrared attenuated total reflection (IR-ATR) spectroscopy has been applied as an analytic tool studying Escherichia coli ( E. coli) biofilm formation close to real time. We report on IR spectroscopic investigations on the biofilm formation via ATR waveguides probing the biofilm in the spectral window of 1800-900 cm-1 at dynamic flow conditions, which facilitated monitoring the growth dynamics during several days. Key IR bands are in the range 1700-1590 cm-1 (amide I), 1580-1490 cm-1 (amide II), and 1141-1006 cm-1 extracellular polymeric substances (EPS), which were evaluated as a function of time. Cyclic fluctuations of the amide I and amide II bands and a continuous increase of the EPS band were related to the starvation of bottom-layered bacteria caused by the nutrient gradient. Potential death of bacteria may then result in cannibalistic behavior known for E. coli colonies. Observing this behavior via IR spectroscopy allows revealing these cyclical changes in bottom-layered bacteria within the biofilm under continuous nutrient flow, in molecular detail, and during extended periods for the first time.

Inhibition of E. coli Growth by Nanodiamond and Graphene Oxide Enhanced by Luria-Bertani Medium.

Nanodiamonds (NDs) and graphene oxide (GO) are modern carbon-based nanomaterials with promising features for the inhibition of microorganism growth ability. Here we compare the effects of nanodiamond and graphene oxide in both annealed (oxidized) and reduced (hydrogenated) forms in two types of cultivation media-Luria-Bertani (LB) and Mueller-Hinton (MH) broths. The comparison shows that the number of colony forming unit (CFU) of Escherichia coli is significantly lowered (45%) by all the nanomaterials in LB medium for at least 24 h against control. On the contrary, a significant long-term inhibition of E. coli growth (by 45%) in the MH medium is provided only by hydrogenated NDs terminated with C-HX groups. The use of salty agars did not enhance the inhibition effects of nanomaterials used, i.e. disruption of bacterial membrane or differences in ionic concentrations do not play any role in bactericidal effects of nanomaterials used. The specific role of the ND and GO on the enhancement of the oxidative stress of bacteria or possible wrapping bacteria by GO nanosheets, therefore isolating them from both the environment and nutrition was suggested. Analyses by infrared spectroscopy, photoelectron spectroscopy, scanning electron microscopy and dynamic light scattering corroborate these conclusions.

Hydroxylation and self-assembly of colloidal hydrogenated nanodiamonds by aqueous oxygen radicals from atmospheric pressure plasma jet.

Plasma chemical surface modification of nanoparticles in gas-liquid type reactors enables a controllable, specific, low-cost, and environmentally friendly alternative to wet chemistry methods or thermal and dry plasma treatments. Here the atmospheric pressure radio-frequency microplasma jet (µ-APPJ) operating with 0.6% O2 in He is used to deliver aqueous oxygen radicals (AOR) to the surface of ∼3 nm hydrogenated detonation nanodiamonds (H-DNDs) suspended in water. The AOR-treated H-DND samples are characterized by FTIR and XPS spectroscopies and by AFM and SEM imaging. The main chemical reaction mechanism is identified as the abstraction of surface hydrogen atoms by O or OH radicals and a consequent attachment of the OH group, thereby increasing concentration of alcohols, carboxyls, and aldehydes on the DND's surface. FTIR spectra reveal also a structural re-arrangement of the surface water on the AOR-treated H-DNDs. Yet zeta-potential of AOR-treated H-DNDs still remains positive (decreases from +45 mV to +30 mV). The chemical modification gives rise to formation of nanoscale chain-like aggregates when AOR-treated H-DNDs are deposited on Si substrate.

Ultrathin Nanocrystalline Diamond Films with Silicon Vacancy Color Centers via Seeding by 2 nm Detonation Nanodiamonds.

Color centers in diamonds have shown excellent potential for applications in quantum information processing, photonics, and biology. Here we report chemical vapor deposition (CVD) growth of nanocrystalline diamond (NCD) films as thin as 5-6 nm with photoluminescence (PL) from silicon-vacancy (SiV) centers at 739 nm. Instead of conventional 4-6 nm detonation nanodiamonds (DNDs), we prepared and employed hydrogenated 2 nm DNDs (zeta potential = +36 mV) to form extremely dense (∼1.3 × 1013 cm-2), thin (2 ± 1 nm), and smooth (RMS roughness < 0.8 nm) nucleation layers on an Si/SiOx substrate, which enabled the CVD growth of such ultrathin NCD films in two different and complementary microwave (MW) CVD systems: (i) focused MW plasma with an ellipsoidal cavity resonator and (ii) pulsed MW plasma with a linear antenna arrangement. Analytical ultracentrifuge, infrared and Raman spectroscopies, atomic force microscopy, and scanning electron microscopy are used for detailed characterization of the 2 nm H-DNDs and the nucleation layer as well as the ultrathin NCD films. We also demonstrate on/off switching of the SiV center PL in the NCD films thinner than 10 nm, which is achieved by changing their surface chemistry.

Uptake and intracellular accumulation of diamond nanoparticles - a metabolic and cytotoxic study.

Diamond nanoparticles, known as nanodiamonds (NDs), possess several medically significant properties. Having a tailorable and easily accessible surface gives them great potential for use in sensing and imaging applications and as a component of cell growth scaffolds. In this work we investigate in vitro interactions of human osteoblast-like SAOS-2 cells with four different groups of NDs, namely high-pressure high-temperature (HPHT) NDs (diameter 18-210 nm, oxygen-terminated), photoluminescent HPHT NDs (diameter 40 nm, oxygen-terminated), detonation NDs (diameter 5 nm, H-terminated), and the same detonation NDs further oxidized by annealing at 450 °C. The influence of the NDs on cell viability and cell count was measured by the mitochondrial metabolic activity test and by counting cells with stained nuclei. The interaction of NDs with cells was monitored by phase contrast live-cell imaging in real time. For both types of oxygen-terminated HPHT NDs, the cell viability and the cell number remained almost the same for concentrations up to 100 µg/mL within the whole range of ND diameters tested. The uptake of hydrogen-terminated detonation NDs caused the viability and the cell number to decrease by 80-85%. The oxidation of the NDs hindered the decrease, but on day 7, a further decrease was observed. While the O-terminated NDs showed mechanical obstruction of cells by agglomerates preventing cell adhesion, migration and division, the H-terminated detonation NDs exhibited rapid penetration into the cells from the beginning of the cultivation period, and also rapid cell congestion and a rapid reduction in viability. These findings are discussed with reference to relevant properties of NDs such as surface chemical bonds, zeta potential and nanoparticle types.