Nanobiophyiscs

We report a Raman band at 1602 cm −1 in the spectra of human cells, which previously had only been observed in mitochondria of yeast cells. This band, which has not yet been assigned to a particular molecular species, was found to occur in HeLa cells, peripheral blood lymphocytes, human mesenchymal stem cells and bovine chondrocytes. The band is proposed as an indicator of the activity of mitochondria in cells. Cells were cultured with and without serum or temporarily deprived of serum. The band can be observed for all these variations in cell culture methodology. The band intensity decreases under the influence of an increase of the calcium ion concentration in the surrounding medium.

A hybrid fluorescence-Raman confocal microscopy platform is presented, which integrates low-wavenumber-resolution Raman imaging, Rayleigh scatter imaging and two-photon fluorescence (TPE) spectral imaging, fast 'amplitude-only' TPE-fluorescence imaging and high-spectral-resolution Raman imaging. This multi-dimensional fluorescence-Raman microscopy platform enables rapid imaging along the fluorescence emission and/or Rayleigh scatter dimensions. It is shown that optical contrast in these images can be used to select an area of interest prior to subsequent investigation with high spatially and spectrally resolved Raman imaging. This new microscopy platform combines the strengths of Raman 'chemical' imaging with light scattering microscopy and fluorescence microscopy and provides new modes of correlative light microscopy. Simultaneous acquisition of TPE hyperspectral fluorescence imaging and Raman imaging illustrates spatial relationships of fluorophores, water, lipid and protein in cells. The fluorescence-Raman microscope is demonstrated in an application to living human bone marrow stromal stem cells.

Activation of hepatic stellate cells has been recognized as one of the first steps in liver injury and repair. During activation, hepatic stellate cells transform into myofibroblasts with concomitant loss of their lipid droplets (LDs) and production of excessive extracellular matrix. Here we aimed to obtain more insight in the dynamics and mechanism of LD loss. We have investigated the LD degradation processes in rat hepatic stellate cells in vitro with a combined approach of confocal Raman microspectroscopy and mass spectrometric analysis of lipids (lipidomics). Upon activation of the hepatic stellate cells, LDs reduce in size, but increase in number during the first 7 days, but the total volume of neutral lipids did not decrease. The LDs also migrate to cellular extensions in the first 7 days, before they disappear. In individual hepatic stellate cells. all LDs have a similar Raman spectrum, suggesting a similar lipid profile. However, Raman studies also showed that the retinyl esters are degraded more rapidly than the triacylglycerols upon activation. Lipidomic analyses confirmed that after 7 days in culture hepatic stellate cells have lost most of their retinyl esters, but not their triacylglycerols and cholesterol esters. Furthermore, we specifically observed a large increase in triacylglycerol-species containing polyunsaturated fatty acids, partly caused by an enhanced incorporation of exogenous arachidonic acid. These results reveal that lipid droplet degradation in activated hepatic stellate cells is a highly dynamic and regulated process. The rapid replacement of retinyl esters by polyunsaturated fatty acids in LDs suggests a role for both lipids or their derivatives like eicosanoids during hepatic stellate cell activation. Citation: Testerink N, Ajat M, Houweling M, Brouwers JF, Pully VV, et al. (2012) Replacement of Retinyl Esters by Polyunsaturated Triacylglycerol Species in Lipid Droplets of Hepatic Stellate Cells during Activation. PLoS ONE 7(4): e34945.

New technology is developed to observe, in situ and in vivo development of tissues from cultured cells . Cells after seeding grow on bioactive degradable scaffolds that provide the physical and chemical cues to guide differentiation and assembly into three dimensional (3D) tissues. The tissue is grown in a bioreactor, where the proper conditions for growth and differentiation are maintained . We have designed and developed a novel microbioreactor in PDMS (Polydimethylsiloxane). In order to assess the growth conditions through out the tissue, the noninvasive, 3D-spatial resolution of confocal Raman imaging is used to measure chemical parameters in the tissue in a label-free manner. We will present first results of a specially designed micro-bioreactor integrated with a hyper spectral Raman microscope.

We present the development of microbioreactors with a sensitive and accurate optical coupling to a confocal Raman microspectrometer. We show that such devices enable in situ and in vitro investigation of cell cultures for tissue engineering by chemically sensitive Raman spectroscopic imaging techniques. The optical resolution of the Raman microspectrometer allows recognition and chemical analysis of subcellular features. Human bone marrow stromal cells (hBMSCs) have been followed after seeding through a phase of early proliferation until typically 21 days later, well after the cells have differentiated to osteoblasts. Long-term perfusion of cells in the dynamic culture conditions was shown to be compatible with experimental optical demands and off-line optical analysis. We show that Raman optical analysis of cells and cellular differentiation in microbioreactors is feasible down to the level of subcellular organelles during development. We conclude that microbioreactors combined with Raman microspectroscopy are a valuable tool to study hBMSC proliferation, differentiation, and development into tissues under in situ and in vitro conditions.

We report a Raman band at 1602 cm −1 in the spectra of human cells, which previously had only been observed in mitochondria of yeast cells. This band, which has not yet been assigned to a particular molecular species, was found to occur in HeLa cells, peripheral blood lymphocytes, human mesenchymal stem cells and bovine chondrocytes. The band is proposed as an indicator of the activity of mitochondria in cells. Cells were cultured with and without serum or temporarily deprived of serum. The band can be observed for all these variations in cell culture methodology. The band intensity decreases under the influence of an increase of the calcium ion concentration in the surrounding medium.

The ability of DNA to self-assemble into one-, two-and three-dimensional nanostructures 1-14 , combined with the precision that is now possible when positioning nanoparticles or proteins 20-24 on DNA scaffolds, provide a promising approach for the self-organization of composite nanostructures . Predicting and controlling the functions that emerge in self-organized biomolecular nanostructures is a major challenge in systems biology, and although a number of innovative examples have been reported , the emergent properties of systems in which enzymes are coupled together have not been fully explored. Here, we report the self-assembly of a DNA scaffold made of DNA strips that include 'hinges' to which biomolecules can be tethered. We attach either two enzymes or a cofactor-enzyme pair to the scaffold, and show that enzyme cascades or cofactor-mediated biocatalysis can proceed effectively; similar processes are not observed in diffusion-controlled homogeneous mixtures of the same components. Furthermore, because the relative position of the two enzymes or the cofactor-enzyme pair is determined by the topology of the DNA scaffold, it is possible to control the reactivity of the system through the design of the individual DNA strips. This method could lead to the self-organization of complex multi-enzyme cascades.

The use of semiconductor quantum dots (QDs) as optical labels for biorecognition events and biocatalytic processes attracts growing interest. While numerous studies reported on the use of the QDs as fluorescent labels, applications of semiconductor QDs as optical probes of dynamic bioprocesses, such as enzymatic transformations, using fluorescence resonance energy transfer (FRET) or photoinduced electron transfer reactions are still scarce. The replication of DNA by polymerase or telomerization of a nucleic acid by telomerase were monitored by the incorporation of a dye into the replica/ telomers associated with QDs and the use of FRET as readout signal. The scission of duplex DNA linked to CdSe QDs by DNase and the hydrolytic cleavage of peptides bound to CdSe QDs were followed by FRET processes. Recently, the activities of tyrosinase and thrombin were analyzed by the tyrosinase-induced generation of quinone residues on amino acid or peptide capping layers associated with CdSe QDs. This resulted in electron-transfer quenching of the QDs. The subsequent hydrolytic cleavage of the peptide by thrombin removed the quencher and recovered the fluorescence of the QDs.

There is a growing interest in using semiconductor quantum dots (QDs) as optical labels for biosensing events. [1] The sizecontrolled fluorescence properties of QDs, [2] the high fluorescence quantum yields of QDs, [3] and their stability against photobleaching makes QDs superior optical labels for multiplexed analysis of antigen-antibody complexes, nucleic acid-DNA hybrids, [6] and other biorecognition complexes. QDs were also applied to monitor biocatalytic transformations using fluorescence resonance energy transfer (FRET) processes. FRET processes between CdSe/ZnS QDs and dye units incorporated into replicated DNA systems or into telomers were used to probe the activities of polymerase and telomerase, respectively. Similarly, FRET reactions were used to monitor the biocatalytic cleavage of peptides by hydrolytic enzymes. [9] Alternatively, electron-transfer quenching of QDs by quinone-functionalized peptides was used to detect the activity of tyrosinase, and the hydrolytic cleavage of the quinone-modified peptide and the restoration of the fluorescence of the QDs were used to probe the activities of tyrosinase and thrombin, respectively. In all of these QD assays for monitoring enzyme activities, it is mandatory to include a quencher (energy or electron-transfer quencher) in the analyzed samples as a reporter unit. Also, for each of the enzymes, a specific assay needs to be developed.

The unique size-controlled photonic properties of semiconductor quantum dots (QDs) find increasing interest for sensing and biosensing. 1 Numerous studies have employed QDs as fluorescent labels for biorecognition events, such as the formation of immunocomplexes 2 or nucleic acid-DNA duplexes. 3 The use of semiconductor QDs as reporter units for biocatalytic processes is, however, scarce, and only few examples demonstrated the application of the photophysical properties of semiconductor QDs to probe biocatalytic reactions. CdS QDs were coupled with biocatalysts, and the resulting photocurrent was employed to follow the enzyme activity. 4 Fluorescence resonance energy transfer (FRET) from CdSe-ZnS QDs to dye units incorporated into replicated DNA or into telomers was used to follow polymerase or telomerase functions. 5 Similarly, the DNase-catalyzed scission of a duplex DNA, consisting of CdSeand dye-tethered hybridized nucleic acids, was probed by following the FRET quenching of the particle in the duplex structure and the regeneration of the QDs' fluorescence upon the cleavage of the DNA. Recently, the activities of proteolytic enzymes were monitored by fluorescence resonance energy transfer within QDpeptide conjugates. Here we wish to report on the monitoring of tyrosinase activity and of thrombin hydrolytic activities by following the fluorescence quenching within CdSe-ZnS QD-peptide conjugates. In contrast to previous studies, we generate by means of tyrosinase the quencher units in the peptide, and we restore the QD fluorescence by the cleavage of the quencher units.

The electrochemical and photoelectrochemical detection of tyrosinase (TR) activity (an indicative marker for melanoma cancer cells) is reported, using Pt nanoparticles (NPs) or CdS NPs as electrocatalytic labels or photoelectrochemical reporter units. The Pt NPs or CdS NPs are modified with a tyrosine methyl ester, (1), capping layer. Oxidation of the capping layer by TR/O 2 yields the respective L-DOPA and dopaquinone products. The reduction of the resulting mixture of products with citric acid yields the L-DOPA derivative,(3), as a single product. The association of the (3)-functionalized Pt NPs or CdS NPs to a boronic acid monolayer-modified electrode enables the electrochemical transduction of TR activity by the Pt-NPs-electrocatalyzed reduction of H 2 O 2 or the photoelectrochemical transduction of TR activity by the generation of photocurrents in the presence of triethanolamine as a sacrificial electron donor. The detection limits for analyzing TR corresponds to 1 U and 0.1 U by the electrochemical and photoelectrochemical methods, respectively. The association of the Pt NPs or CdS NPs to the functionalized monolayer electrode is followed by quartz crystal microbalance measurements.

Silver nanoparticle aggregates have been shown to support very large enhancements of fluorescence intensity from organic dye molecules coupled with an extreme reduction in observed fluorescence lifetimes. Here we show that for the same type of aggregates, similar enhancement factors (B75Â in intensity and B3400Â in lifetime compared to the native radiative lifetime) are observed for a ruthenium-based phosphorescent dye (when taking into account the effect of charge and the excitation/emission wavelengths). Additionally, the inherently long native phosphorescence lifetimes practically enable more detailed analyses of the distribution of lifetimes (compared with the case with fluorescence decays). It was thus possible to unambiguously observe the deviation from monoexponential decay which we attribute to emission from a distribution of fluorophores with different lifetimes, as we could expect from a random aggregation process. We believe that combining phosphorescent dyes with plasmonic structures, even down to the single dye level, will offer a convenient approach to better characterize plasmonic systems in detail.

Surface-modifi ed quantum dots (QDs) are extensively used as fl uorescent labels for sensing and biosensing events. [ 1 , 2 ] Fluorescence resonance energy transfer (FRET) or electron-transfer quenching of the QDs are two fundamental photophysical mechanisms to probe sensing processes at the surface of the QDs. [ 2 , 3 ] Indeed, these photophysical mechanisms were implemented to detect ions, [ 4 ] to follow pH changes, [ 5 ] and to detect molecular substrates, such as saccharides or neurotransmitters. [ 6 ] Similarly, QDs have been widely used for biosensing, and different luminescence sensors that follow the formation of antigen-antibody complexes, [ 7 ] DNA hybridization [ 8 , 9 ] and aptamer-substrate complexes [ 10 ] were reported. Also, QDs have been extensively used to follow biocatalytic transformations and to quantitatively probe the substrates of enzymes. [ 9 , 11 ] Here we wish to report on the use of chemically modifi ed CdSe/ZnS QDs as fl uorescent probes for the analysis of explosives, and specifi cally, the detection of trinitrotoluene (TNT) or trinitrotriazine (RDX). The QDs are functionalized with electron donating ligands that bind nitro-containing explosives, exhibiting electron acceptor properties, to the QDs surfaces, via supramolecular donor-acceptor interactions. The concentration of the explosive substrates at the QDs via the donor-acceptor complexes leads to the quenching of the luminescence of the QDs, thus allowing the quantitative analysis of the substrates. The sensitivities of the QDs sensors are controlled by the electron donating properties of the capping layer that modifi es the particles. The rapid and sensitive analysis of nitroaromatic explosives attracts substantial recent efforts, and different optical [ 12 ] or electrochemical [ 13 ] TNT or RDX sensors were reported. Other sensors for explosives include surface acoustic wave (SAW) systems, where piezoelectric crystal coated with absorbing materials such as carbowax [ 14 ] or cyclodextrin polymers, [ 15 ] were

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0 × 10 −14 M without the need for a spectrometer or a scanning stage.

We introduce a sensing platform for specific detection of DNA based on the formation of gold nanoparticles dimers on a surface. The specific coupling of a second gold nanoparticle to a surface bound nanoparticle by DNA hybridization results in a red shift of the nanoparticle plasmon peak. This shift can be detected as a color change in the darkfield image of the gold nanoparticles. Parallel detection of hundreds of gold nanoparticles with a calibrated true color camera enabled us to detect specific binding of target DNA. This enables a limit of detection below 1.0 × 10 −14 M without the need for a spectrometer or a scanning stage.