Isotope labeled 3D-Raman confocal imaging and atomic force microscopy study on epithelial cells interacting with the fungus Candida albicans.

The human pathogenic fungus Candida albicans damages epithelial cells during superficial infections. Here we use three-dimensional-sequential-confocal Raman spectroscopic imaging and atomic force microscopy to investigate the interaction of C. albicans wild type cells, the secreted C. albicans peptide toxin candidalysin and mutant cells lacking candidalysin with epithelial cells. The candidalysin is responsible for epithelial cell damage and exhibits in its deuterated form an identifiable Raman signal in a frequency region distinct from the cellular frequency region. Vibration modes at 2100-2200 cm-1 attributed to carbon­deuterium bending and at 477 cm-1, attributed to the nitrogen­deuterium out-of-plane bending, found around the nucleus, can be assigned to deuterated candidalysin. Atomic force microscopy visualized 100 nm deep lesions on the cell and force-distance curves indicate the higher adhesion on pore surrounding after incubation with candidalysin. Candidalysin targets the plasma membrane, but is also found inside of the cytosol of epithelial cells during C. albicans infection.

Electrochemical growth mechanism of nanoporous platinum layers.

Porous platinum is a frequently used catalyst material in electrosynthesis and a robust broadband absorber in thermoelectrics. Pore size distribution and localization determine its properties by a large extent. However, the pore formation mechanism during the growth of the material remains unclear. In this work we elucidate the mechanism underlying electrochemical growth of nanoporous platinum layers and its control by ionic concentration and current density during electrolysis. The electrode kinetics and reduction steps of PtCl4 on platinum electrodes are investigated by cyclic voltammetry and impedance measurements. Cyclic voltammograms show three reduction steps: two steps relate to the platinum cation reduction, and one step relates to the hydrogen reduction. Hydrogen is not involved in the reduction of PtCl4, however it enables the formation of nanopores in the layers. These findings contribute to the understanding of electrochemical growth of nanoporous platinum layers in isopropanol with thickness of 100 nm to 500 nm.

Multi-wavelength, handheld laser speckle imaging for skin evaluation.

OBJECTIVE: A handheld device was developed and qualified for in vivo human skin evaluation using laser speckle imaging technology. METHODS: Each laser speckle device prototype allows the choice of up to three different laser wavelengths in the range of 400 nm to 800 nm in total. Speckle pattern analysis gives various speckle parameters, for example, speckle contrast, speckle size, speckle modulation or fractal dimension. The developed laser speckle device prototypes were evaluated investigating three skin issues. RESULTS: We receive reproducible results from the speckle imaging device. For skin ageing, we found significant changes within three age groups. The effect of a methyl nicotinate treatment was clearly visible and quantifiable using a moorFLPI device as well as our speckle imaging device. In terms of basal cell carcinoma diagnosis, we found significant differences between normal and diseased skin, even though the number of samples was limited. CONCLUSION: As shown with first application examples, it was possible to demonstrate the potential of the method for skin evaluation in vivo.

Electro-architected porous platinum on metallic multijunction nanolayers to optimize their optical properties for infrared sensor application.

Tailoring the physicochemical properties of the metallic multijunction nanolayers is a prerequisite for the development of microelectronics. From this perspective, a desired lower reflectance of infrared radiation was achieved by an electrochemical deposition of porous platinum in nonaqueous media on silver mirror supported nickel-chrome and nickel-titanium metallic films with incremental decreasing thicknesses from 80-10 nm. The electro-assembled architectures were examined by means of scanning electron microscopy and Fourier transform infrared spectroscopy and it was observed that the layer and sublayer thicknesses and resistivities have a substantial effect upon the porous platinum morphology and its optical properties. It is here reported that the augmentation of the metallic layer electrical conductivity determines the electroformation of more compact platinum nanolayers. Moreover, the platinum black coating of metallic nanolayers causes a considerable decrease of the reflectance in the region from 1000-8000 cm-1.

Surface-enhanced Raman spectroscopy of cell lysates mixed with silver nanoparticles for tumor classification.

The throughput of spontaneous Raman spectroscopy for cell identification applications is limited to the range of one cell per second because of the relatively low sensitivity. Surface-enhanced Raman scattering (SERS) is a widespread way to amplify the intensity of Raman signals by several orders of magnitude and, consequently, to improve the sensitivity and throughput. SERS protocols using immuno-functionalized nanoparticles turned out to be challenging for cell identification because they require complex preparation procedures. Here, a new SERS strategy is presented for cell classification using non-functionalized silver nanoparticles and potassium chloride to induce aggregation. To demonstrate the principle, cell lysates were prepared by ultrasonication that disrupts the cell membrane and enables interaction of released cellular biomolecules to nanoparticles. This approach was applied to distinguish four cell lines - Capan-1, HepG2, Sk-Hep1 and MCF-7 - using SERS at 785 nm excitation. Six independent batches were prepared per cell line to check the reproducibility. Principal component analysis was applied for data reduction and assessment of spectral variations that were assigned to proteins, nucleotides and carbohydrates. Four principal components were selected as input for classification models based on support vector machines. Leave-three-batches-out cross validation recognized four cell lines with sensitivities, specificities and accuracies above 96%. We conclude that this reproducible and specific SERS approach offers prospects for cell identification using easily preparable silver nanoparticles.

Chemo-spectroscopic sensor for carboxyl terminus overexpressed in carcinoma cell membrane.

Certain carboxyl groups of the plasma membrane are involved in tumorgenesis processes. A gold core-hydroxyapatite shell (AuHA) nanocomposite is introduced as chemo-spectroscopic sensor to monitor these carboxyl groups of the cell membrane. Hydroxyapatite (HA) plays the role both of a chemical detector and of a biocompatible Raman marker. The principle of detection is based on chemical interaction between the hydroxyl groups of the HA and the carboxyl terminus of the proteins. The AuHA exhibits a surface enhanced Raman scattering (SERS) signal at 954 cm(-1) which can be used for its localization. The bio-sensing capacity of AuHA towards human skin epidermoid carcinoma (A431) and Chinese hamster ovary (CHO) cell lines is investigated using Raman microspectroscopic imaging. The localization of AuHA on cells is correlated with scanning electron microscopy, transmission electron microscopy and structured illumination fluorescence microscopy. This qualitative approach is a step towards a quantitative study of the proteins terminus. FROM THE CLINICAL EDITOR: This method would enable further studies on the molecular profiling of the plasma membrane, in an attempt to provide accurate cell identification. Using a gold core-hydroxyapatite shell (AuHA) nanocomposite, the authors in this paper showed the feasibility of detecting and differentiating cell surface molecules by surface enhanced Raman scattering.

Magnetic apatite for structural insights on the plasma membrane.

The iron oxide-hydroxyapatite (FeOxHA) nanoparticles reported here differ from those reported before by their advantage of homogeneity and simple preparation; moreover, the presence of carboxymethyldextran (CMD), together with hydroxyapatite (HA), allows access to the cellular membrane, which makes our magnetic apatite unique. These nanoparticles combine magnetic behavior, Raman label ability and the property of interaction with the cellular membrane; they therefore represent an interesting material for structural differentiation of the cell membrane. It was observed by Raman spectroscopy, scanning electron microscopy (SEM) and fluorescence microscopy that FeOxHA adheres to the plasma membrane and does not penetrate the membrane. These insights make the nanoparticles a promising material for magnetic cell sorting, e.g. in microfluidic device applications.

Labeling of the pathogenic bacterium Staphylococcus aureus with gold or ferric oxide-core nanoparticles highlights new capabilities for investigation of host-pathogen interactions.

Throughout the world, infections caused by bacteria such as Staphylococcus aureus are a major cause of morbidity and mortality. In order to gain some understanding of the complicated physiological link between host and pathogen, modern techniques such as confocal microscopy and sophisticated OMICs technologies are suitable. However, labeling of pathogens such as S. aureus with green fluorescent protein, for example, or the generation of a reliable antibody, which are prerequisites for the application of reproducible isolation techniques, does not always succeed. Here, we present a universal approach for monitoring pathogen traffic after internalization into host cells by fluorescence microscopy and for isolation of bacteria from host-pathogen interaction assays using gold or ferric oxide-core, poly(vinyl alcohol) coated, and fluorescence-labeled nanoparticles (NP). The incubation of S. aureus HG001 with those NP had only minor effects on the bacterial growth in vitro. Quantitative proteome analysis after 24 h of NP incubation revealed that presence of NP provoked only marginal changes in the proteome pattern. The method presented enabled us to investigate the behavior of S. aureus HG001 during infection of S9 human epithelial cells by means of fluorescence microscopy and proteomics using magnetic separation or cell sorting.

Raman-on-chip device and detection fibres with fibre Bragg grating for analysis of solutions and particles.

An all-fibre based Raman-on-chip setup is introduced which enables analysis of solutions and trapped particles without microscopes or objectives. Beside the novel quartz microfluidic chip, innovative multi-core single-mode fibres with integrated fibre Bragg gratings are used for detection. The limit of quantitation is 7.5 mM for urea and 2.5 mM for nicotine with linear Raman spectroscopy. This is an improvement of more than two orders of magnitude compared with previous fibre-based microfluidic Raman detection schemes. Furthermore, our device was combined with optical traps to collect Raman-on-chip spectra of spherical polymer beads.

Uptake of well-defined, highly glycosylated, pentafluorostyrene-based polymers and nanoparticles by human hepatocellular carcinoma cells.

Chain length, size, composition, surface charge, and other properties of polymeric materials affect their recognition and uptake by cells and must be optimized to deliver polymers selectively to their target. However, it is often not possible to precisely modify selected properties without changing other parameters. To overcome these difficulties, well-defined poly(pentafluorostyrene)-based polymers are prepared that can be grafted via thiol/para-fluorine "click" reaction with 1-thio-ß-D-glucose and 1-thio-ß-D-galactose. Fluorescence microscopy and flow cytometry show that nanoparticles are taken up by HepG2 cells to a higher degree than the respective water-soluble polymers, and that internalization of both galactosylated homo- and nanoprecipitated block copolymers is enhanced.

Simultaneous topographic and amperometric membrane mapping using an AFM probe integrated biosensor.

The investigation of the plasma membrane with intercorrelated multiparameter techniques is a prerequisite for understanding its function. Presented here, is a simultaneous electrochemical and topographic study of the cell membrane using a miniaturized amperometric enzymatic biosensor. The fabrication of this biosensor is also reported. The biosensor combines a scanning force microscopy (AFM) gold-coated cantilever and an enzymatic transducer layer of peroxidases (PODs). When these enzymes are brought in contact with the substrate, the specific redox reaction produces an electric current. The intensity of this current is detected simultaneously with the surface imaging. For sensor characterization, hydroquinone-2-carboxylic acid (HQ) is selected as an intrinsic source of H(2)O(2). HQ has been electrochemically regenerated by the reduction of antraquinone-2-carboxylic acid (AQ). The biosensor reaches the steady state value of the current intensity in 1 ± 0.2s.

Development and critical evaluation of fluorescent chloride nanosensors.

In this study, we describe the preparation and evaluation of new fluorescent sensor nanoparticles for the ratiometric measurement of chloride concentrations. Both a chloride-sensitive dye (lucigenin) and a reference dye (sulforhodamine derivative) were incorporated into polyacrylamide nanoparticles via inverse microemulsion polymerization and investigated for their response to chloride ions in buffered suspension as well as in living cells. The fluorescence intensity of lucigenin reversibly decreased in the presence of chloride ions due to a collisional quenching process, which can be described with the Stern-Volmer equation. The determined Stern-Volmer constant K SV for the quenching of lucigenin incorporated into particles was found to be 53 M (-1) and is considerably smaller than the Stern-Volmer constant for quenching of free lucigenin ( K SV = 250 M (-1)) under the same conditions. To test the nanosensors in living cells, we incorporated them into Chinese hamster ovary cells and mouse fibroblasts by using the conventional lipofectamin technique and monitored the response to changing chloride concentrations in the cell.

A flexible method for the fabrication of gold nanostructures using oligonucleotide derivatives.

Several linear and branched DNA structures from 80-200 nm with a biotine molecule in the middle have been prepared. These structures have been decorated by addition of positively charged gold nanoparticles carrying 4-(dimethylamino)pyridine ligands. Streptavidin binds to the central biotine molecule introducing a 20 nm gap in the structure in which a biotinylated nanoparticle can be introduced. The simplest structure (80 nm, linear) is formed by 4 oligonucleotides. By changing some of these components changes on length, shape, and recognition system easily can be introduced.

DNA-templated assembly of nanoscale architectures for next-generation electronic devices.

We report the assembly and structural characterization of a Y-shaped DNA template incorporating a central biotin moiety. We also report that this template may be used assemble nanoscale architectures, which demonstrate the potential of this and related approaches to the fabrication of next-generation electronic devices. Of particular significance is the finding that it is possible to selectively metallize the above DNA template to obtain a three-electrode configuration. Also of particular significance is finding that a biotin modified nanoparticle will recognize and bind selectively the central biotin moiety of the same template, once functionalized by the protein streptavidin.

Phenols monitoring and Hill coefficient evaluation using tyrosinase-based amperometric biosensors.

Sensitive amperometric biosensors for phenols compounds, based on tyrosinase (polyphenoloxidase, PPO) immobilized on a Pt electrode in an electropolymerized poly-amphiphilic pyrrole matrix or cross-linked with glutaraldehyde, were constructed and compared. Steady-state amperometric measurements, performed at -50 mV vs. SCE in aqueous phosphate buffer containing LiClO(4) 0.1 M (pH 7) as well as in a chloroform solution containing 0.1 M C(6)H(5)CH(2)N(CH(3))(3)Cl, were used in order to compare the electroanalytical and kinetic parameters of the investigated amperometric biosensors in aqueous and nonaqueous media. It was established that the polypyrrole matrix has a higher efficiency for enzyme retention resulting in higher bioelectrode sensitivity, both in aqueous buffer (690 microA M(-1)) and in chloroform (149 microA M(-1)).

Biosensors for phenol derivatives using biochemical signal amplification.

Two different approaches, both exploiting two enzymes cooperative functioning, to enhance the sensitivity of tyrosinase (PPO) based biosensor for amperometric detection of phenols have been compared. For this purpose, one monoenzyme electrode (PPO) and two bienzyme electrodes (PPO and d-glucose dehydrogenase, GDH; PPO and horseradish peroxidase, HRP) were constructed using agar-agar gel as enzyme immobilization matrix. The biosensors responses for l-tyrosine detection were recorded at -50 mV versus saturated calomel electrode (SCE). The highest sensitivity (74 mA M(-1)) was observed for the PPO-GDH couple, while that recorded for PPO-HRP couple system was only 32 times higher than that measured for monoenzyme electrode (0.01 mA M(-1)). The ability of the PPO-, PPO-GDH-, PPO-HRP-based biosensors to assay phenols was demonstrated by quantitative determination of phenol, 1,2-dihydroxybenzene, 1,3-dihydroxybenzene, 1,4-dihydroxybenzene, 2-amino-3 (4-hydroxyphenyl) propanoic acid, 2-hydroxytoluene, 3-hydroxytoluene, 4-hydroxytoluene, 4-clorophenol, 3-clorophenol, 2-clorophenol, 4-hydroxybenzoic acid.