Atomistic Picture of Fluorescent Probes with Hydrocarbon Tails in Lipid Bilayer Membranes: An Investigation of Selective Affinities and Fluorescent Anisotropies in Different Environmental Phases.

By reverting to spectroscopy, changes in the biological environment of a fluorescent probe can be monitored and the presence of various phases of the surrounding lipid bilayer membranes can be detected. However, it is currently not always clear in which phase the probe resides. The well-known orange 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbo-cyanine perchlorate (DiI-C18(5)) fluorophore, for instance, and the new, blue BODIPY (4,4-difluoro-4-bora-3 a,4 a-diaza- s-indacene) derivative were experimentally seen to target and highlight identical parts of giant unilamellar vesicles of various compositions, comprising mixtures of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), sphingomyelin (SM), and cholesterol (Chol). However, it was not clear which of the coexisting membrane phases were visualized (Bacalum et al., Langmuir. 2016, 32, 3495). The present study addresses this issue by utilizing large-scale molecular dynamics simulations and the z-constraint method, which allows evaluating Gibbs free-energy profiles. The current calculations give an indication why, at room temperature, both BODIPY and DiI-C18(5) probes prefer the gel (So) phase in DOPC/DPPC (2:3 molar ratio) and the liquid-ordered (Lo) phase in DOPC/SM/Chol (1:2:1 molar ratio) mixtures. This study highlights the important differences in orientation and location and therefore in efficiency between the probes when they are used in fluorescence microscopy to screen various lipid bilayer membrane phases. Dependent on the lipid composition, the angle between the transition-state dipole moments of both probes and the normal to the membrane is found to deviate clearly from 90°. It is seen that the DiI-C18(5) probe is located in the headgroup region of the SM/Chol mixture, in close contact with water molecules. A fluorescence anisotropy study also indicates that DiI-C18(5) gives rise to a distinctive behavior in the SM/Chol membrane compared to the other considered membranes. The latter behavior has not been seen for the studied BODIPY probe, which is located deeper in the membrane.

The role of chiral local field enhancements below the resolution limit of Second Harmonic Generation microscopy.

While it has been demonstrated that, above its resolution limit, Second Harmonic Generation (SHG) microscopy can map chiral local field enhancements, below that limit, structural defects were found to play a major role. Here we show that, even below the resolution limit, the contributions from chiral local field enhancements to the SHG signal can dominate over those by structural defects. We report highly homogeneous SHG micrographs of star-shaped gold nanostructures, where the SHG circular dichroism effect is clearly visible from virtually every single nanostructure. Most likely, size and geometry determine the dominant contributions to the SHG signal in nanostructured systems.

Hotspot decorations map plasmonic patterns with the resolution of scanning probe techniques.

In high definition mapping of the plasmonic patterns on the surfaces of nanostructures, the diffraction limit of light remains an important obstacle. Here we demonstrate that this diffraction limit can be completely circumvented. We show that upon illuminating nanostructures made of nickel and palladium, the resulting surface-plasmon pattern is imprinted on the structures themselves; the hotspots (regions of local field enhancement) are decorated with overgrowths, allowing for their subsequent imaging with scanning-probe techniques. The resulting resolution of plasmon pattern imaging is correspondingly improved.

Linearly polarized second harmonic generation microscopy reveals chirality.

In optics, chirality is typically associated with circularly polarized light. Here we present a novel way to detect the handedness of chiral materials with linearly polarized light. We performed Second Harmonic Generation (SHG) microscopy on G-shaped planar chiral nanostructures made of gold. The SHG response originates in distinctive hotspots, whose arrangement is dependent of the handedness. These results uncover new directions for studying chirality in artificial materials.

Plasmonic ratchet wheels: switching circular dichroism by arranging chiral nanostructures.

We demonstrate circular dichroism (CD) in the second harmonic generation (SHG) signal from chiral assemblies of G-shaped nanostructures made of gold. The arrangement of the G shapes is crucial since upon reordering them the SHG-CD effect disappears. Microscopy reveals SHG "hotspots" assemblies, which originate in enantiomerically sensitive plasmon modes, having the novel property of exhibiting a chiral geometry themselves in relation with the handedness of the material. These results open new frontiers in studying chirality.

Molecular understanding of label-free second harmonic imaging of microtubules.

Microtubules are a vital component of the cell's cytoskeleton and their organization is crucial for healthy cell functioning. The use of label-free SH imaging of microtubules remains limited, as sensitive detection is required and the true molecular origin and main determinants required to generate SH from microtubules are not fully understood. Using advanced correlative imaging techniques, we identified the determinants of the microtubule-dependent SH signal. Microtubule polarity, number and organization determine SH signal intensity in biological samples. At the molecular level, we show that the GTP-bound tubulin dimer conformation is fundamental for microtubules to generate detectable SH signals. We show that SH imaging can be used to study the effects of microtubule-targeting drugs and proteins and to detect changes in tubulin conformations during neuronal maturation. Our data provide a means to interpret and use SH imaging to monitor changes in the microtubule network in a label-free manner.

A MIP-based impedimetric sensor for the detection of low-MW molecules.

Mimicking the selectivity and sensitivity of biological systems for sensor devices is of increasing interest in biomedical, environmental and chemical analysis. Synthetic materials with imprinted nanocavities, acting as highly selective artificial receptors, are a tailor-made solution in obtaining such a sensor. Incorporation of such molecularly imprinted polymers (MIPs) in a platform suitable for electrochemical measurements, can offer high sensitivity together with device miniaturization and an electronic read-out. As a proof of principle, a MIP-based sensor for L-nicotine has been developed. To this end, the molecular structure of L-nicotine was imprinted in a polymer matrix of polymethacrylic acid (PMAA). Subsequently, microparticles of the imprinted polymer were immobilized on thin films of the conjugated polymer OC(1)C(10)-PPV. These films were incorporated in an impedimetric sensing device. Using electrochemical impedance spectroscopy, the real part of the impedance was monitored for various concentrations. This setup allows for the detection of l-nicotine from 1 to 10 nM and is insensitive for the resembling molecule L-cotinine.

EDC-mediated DNA attachment to nanocrystalline CVD diamond films.

Chemical vapour deposited (CVD) diamond is a very promising material for biosensor fabrication owing both to its chemical inertness and the ability to make it electrical semiconducting that allows for connection with integrated circuits. For biosensor construction, a biochemical method to immobilize nucleic acids to a diamond surface has been developed. Nanocrystalline diamond is grown using microwave plasma-enhanced chemical vapour deposition (MPECVD). After hydrogenation of the surface, 10-undecenoic acid, an omega-unsaturated fatty acid, is tethered by 254 nm photochemical attachment. This is followed by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC)-mediated attachment of amino (NH(2))-modified dsDNA. The functionality of the covalently bound dsDNA molecules is confirmed by fluorescence measurements, PCR and gel electrophoresis during 35 denaturation and rehybridisation steps. The linking method after the fatty acid attachment can easily be applied to other biomolecules like antibodies and enzymes.

Impedimetric immunosensors based on the conjugated polymer PPV.

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.

Simultaneous analysis of multiple fluorescence decay curves by Laplace transforms. Deconvolution with reference or excitation profiles.

The properties and potentials of the noniterative Laplace deconvolution (LAP2) (M. Ameloot and H. Hendrickx, Biophys. J. 44 (1983) 27) are further investigated. It is shown that LAP2 is exact and that no extrapolations have to be calculated or assumed for the data measured in the actual time window if the impulse response function of the investigated system can be described by a sum of exponentials. The formulas for the LAP2 deconvolution against the measured decay of a reference compound instead of the recorded excitation profile are derived. The procedure for the simultaneous analysis of multiple fluorescence decay curves by LAP2 is described in detail. This global analysis allows one to link any decay parameter, is fast and compares favorably with the nonlinear least-squares iterative reconvolution methods. Because of its short computation time the global analysis by LAP2 provides an efficient way to analyze the fluorescence decay surface in terms of decay associated spectra.

Nonlinear superchiral meta-surfaces: tuning chirality and disentangling non-reciprocity at the nanoscale.

Circularly polarized light is incident on a nanostructured chiral meta-surface. In the nanostructured unit cells whose chirality matches that of light, superchiral light is forming and strong optical second harmonic generation can be observed.

Rapid assessment of the stability of DNA duplexes by impedimetric real-time monitoring of chemically induced denaturation.

In this article, we report on the electronic monitoring of DNA denaturation by NaOH using electrochemical impedance spectroscopy in combination with fluorescence imaging as a reference technique. The probe DNA consisting of a 36-mer fragment was covalently immobilized on nanocrystalline-diamond electrodes and hybridized with different types of 29-mer target DNA (complementary, single-nucleotide defects at two different positions, and a non-complementary random sequence). The mathematical separation of the impedimetric signals into the time constant for NaOH exposure and the intrinsic denaturation-time constants gives clear evidence that the denaturation times reflect the intrinsic stability of the DNA duplexes. The intrinsic time constants correlate with calculated DNA-melting temperatures. The impedimetric method requires minimal instrumentation, is label-free and fast with a typical time scale of minutes and is highly reproducible. The sensor electrodes can be used repetitively. These elements suggest that the monitoring of chemically induced denaturation at room temperature is an interesting approach to measure DNA duplex stability as an alternative to thermal denaturation at elevated temperatures, used in DNA-melting experiments and single nucleotide polymorphism (SNP) analysis.

Na-P(i) cotransporter type I activity causes a transient intracellular alkalinization during ATP depletion in rabbit medullary thick ascending limb cells.

The cellular pathophysiology of renal ischemia-reperfusion injury was investigated in primary cell cultures from rabbit medullary thick ascending limb (MTAL). Metabolic inhibition (MI) was achieved with cyanide and 2-deoxyglucose. Sixty minutes of MI caused a profound but reversible decrease in intracellular concentration of ATP ([ATP]i). Intracellular pH (pHi) first decreased after initiation of MI, followed by a transient alkalinization. When [ATP]i reached its lowest value (<1% of control), the cells slowly acidified to reach a stable pHi of 6.92 after 50 min of MI. In the presence of EIPA (10 micromol/L), the pattern of changes in pHi was unchanged and acidification was not increased, indicating that the Na+/H+ exchangers were inactive during ATP depletion. When inorganic phosphate (P(i)) or Na+ was omitted from the apical solutions during MI, the transient alkalinization was no longer observed and the cytosol slowly acidified. Experiments on Na+-dependent alkalinizations revealed the presence of a Na-P(i) cotransporter in the apical cell membrane. With indirect immunofluorescence, the Na-P(i) cotransporter expressed in these primary cell cultures could be identified as Na-P(i) type I. Although the exact physiological role of Na-P(i) type I still is unresolved, these experiments demonstrate that apical Na-P(i) type I activity is increased at the onset of ATP depletion in MTAL cells.

Measurement of cytosolic and mitochondrial pH in living cells during reversible metabolic inhibition.

Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.

Topographical and functional characterization of the ssDNA probe layer generated through EDC-mediated covalent attachment to nanocrystalline diamond using fluorescence microscopy.

The covalent attachment method for DNA on nanocrystalline diamond (NCD), involving the introduction of COOH functionalities on the surface by photoattachment of 10-undecenoic acid (10-UDA), followed by the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)-mediated coupling to NH 2-labeled ssDNA, is evaluated in terms of stability, density, and functionality of the resulting biological interface. This is of crucial importance in DNA biosensor development. The covalent nature of DNA attachment will infer the necessary stability and favorable orientation to the ssDNA probe molecules. Using confocal fluorescence microscopy, the influence of buffer type for the removal of excess 10-UDA and ssDNA, the probe ssDNA length, the probe ssDNA concentration, and the presence of the COOH-linker on the density and functionality of the ssDNA probe layer were investigated. It was determined that the most homogeneously dense and functional DNA layer was obtained when 300 pmol of short ssDNA was applied to COOH-modified NCD samples, while H-terminated NCD was resistant for DNA attachment. Exploiting this surface functionality dependence of the DNA attachment efficiency, a shadow mask was applied during the photochemical introduction of the COOH-functionalities, leaving certain regions on the NCD H-terminated. The subsequent DNA attachment resulted in a fluorescence pattern corresponding to the negative of the shadow mask. Finally, NCD surfaces covered with mixtures of the 10-UDA linker molecule and a similar molecule lacking the COOH functionality, functioning as a lateral spacer, were examined for their suitability in preventing nonspecific adsorption to the surface and in decreasing steric hindrance. However, purely COOH-modified NCD samples, patterned with H-terminated regions and treated with a controlled amount of probe DNA, proved the most efficient in fulfilling these tasks.

Towards a real-time, label-free, diamond-based DNA sensor.

Most challenging in the development of DNA sensors is the ability to distinguish between fully complementary target ssDNA (single-strand DNA) and 1-mismatch ssDNA. To deal with this problem, we performed impedance spectroscopy on DNA-functionalized nanocrystalline diamond (NCD) layers during hybridization and denaturation. In both reactions, a difference in behavior was observed for 1-mismatch target DNA and complementary target DNA in real-time. During real-time hybridization, a decrease of the impedance was observed at lower frequencies when the complementary target DNA was added, while the addition of 1-mismatch target ssDNA caused no significant change. Fitting these results to an electrical circuit demonstrates that this is correlated with a decrease of the depletion zone in the space charge region of the diamond. During real-time denaturation, differentiation between 1-mismatch and complementary target DNA was possible at higher frequencies. Denaturation of complementary DNA showed the longest exponential decay time of the impedance, while the decay time during 1-mismatch denaturation was the shortest. The real-time hybridization and denaturation experiments were carried out on different NCD samples in various buffer solutions at temperatures between 20 and 80 degrees C. It was revealed that the best results were obtained using a Microhyb hybridization buffer at 80 degrees C and 10x PCR buffer at 30 degrees C for hybridization and 0.1 M NaOH at temperatures above 40 degrees C for denaturation. We demonstrate that the combination of real-time hybridization spectra and real-time denaturation spectra yield important information on the type of target. This approach may allow a reliable identification of the mismatch sequence, which is the most biologically relevant.