Interaction of fluorescent molecular rotors with blood plasma proteins

Many disease states have associated blood viscosity changes. Molecular rotors, fluorescent molecules with viscosity sensitive quantum yields, have recently been investigated as a new method for biofluid viscosity measurement. Current viscometer measurements are complicated by proteins adhering to surfaces and forming air-surface layers. It is unknown at this time what effects proteins may have on biofluid viscosity measurements using molecular rotors. To answer this question, binding affinities to blood plasma proteins were investigated by equilibrium dialysis for four hydrophilic molecular rotors. Aqueous solutions of 9-[(2-cyano-2-hydroxy-carbonyl)vinyl]julolidine (CCVJ) and three derivatives were prepared and dialyzed against solutions of bovine source albumin, fibrinogen and immunoglobulin G approximating normal physiologic concentrations and fresh-frozen human plasma. After equilibration, dye concentration on each side of the dialysis membrane was assessed by spectrophotometry. The relative binding affinity of the four dyes to the proteins and to the plasma was compared. Affinity of all dyes was highest for albumin. The bound dye fraction showed little change in relation to protein concentration in the physiological concentration range. Diol, the most hydrophilic molecular rotor tested showed the lowest affinity for albumin. This study indicates that hydrophilic molecular rotors are well-suited for biofluid viscosity measurement.     

Precision assessment of biofluid viscosity measurements using molecular rotors

Blood viscosity changes with many pathologic conditions, but its importance has not been fully investigated because the current methods of measurement are poorly suited for clinical applications. The use of viscosity-sensitive fluorescent molecular rotors to determine fluid viscosity in a nonmechanical manner has been investigated recently, but it is unknown how the precision of the fluorescence-based method compares to established mechanical viscometry. Human blood plasma viscosity was modulated with high-viscosity plasma expanders, dextran, pentastarch, and hetastarch. The samples were divided into a calibration and a test set. The relationship between fluorescence emission and viscosity was established using the calibration set. Viscosity of the test set was determined by fluorescence and by cone-and-plate viscometer, and the precision of both methods compared. Molecular rotor fluorescence intensity showed a power law relationship with solution viscosity. Mechanical measurements deviated from the theoretical viscosity value by less than 7.6%, while fluorescence-based measurements deviated by less than 6%. The average coefficient of variation was 6.9% (mechanical measurement) and 3.4% to 3.8% (fluorescence-based measurement, depending on the molecular rotor used). Fluorescence-based viscometry exhibits comparable precision to mechanical viscometry. Fluorescence viscometry does not apply shear and is therefore more practical for biofluids which have apparent non-Newtonian properties. In addition, fluorescence instrumentation makes very fast serial measurements possible, thus promising new areas of application in laboratory and clinical settings.     

Environment-sensitive behavior of fluorescent molecular rotors

Molecular rotors are a group of fluorescent molecules that form twisted intramolecular charge transfer (TICT) states upon photoexcitation. When intramolecular twisting occurs, the molecular rotor returns to the ground state either by emission of a red-shifted emission band or by nonradiative relaxation. The emission properties are strongly solvent-dependent, and the solvent viscosity is the primary determinant of the fluorescent quantum yield from the planar (non-twisted) conformation. This viscosity-sensitive behavior gives rise to applications in, for example, fluid mechanics, polymer chemistry, cell physiology, and the food sciences. However, the relationship between bulk viscosity and the molecular-scale interaction of a molecular rotor with its environment are not fully understood. This review presents the pertinent theories of the rotor-solvent interaction on the molecular level and how this interaction leads to the viscosity-sensitive behavior. Furthermore, current applications of molecular rotors as microviscosity sensors are reviewed, and engineering aspects are presented on how measurement accuracy and precision can be improved.     

Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes

Fluorescent molecular rotors belong to a group of twisted intramolecular charge transfer complexes (TICT) whose photophysical characteristics depend on their environment. In this study, the influence of solvent polarity and viscosity on several representative TICT compounds (three Coumarin derivatives, 4,4-dimethylaminobenzonitrile DMABN, 9-(dicyanovinyl)-julolidine DCVJ), was examined. While solvent polarity caused a bathochromic shift of peak emission in all compounds, this shift was lowest in the case of molecular rotors. Peak intensity was influenced strongly by solvent viscosity in DMABN and the molecular rotors, but polarity and viscosity influences cannot be separated with DMABN. Coumarins, on the other hand, did not show viscosity sensitivity. This study shows the unique suitability of molecular rotors as fluorescent viscosity sensors.     

Coumarin-like fluorescent molecular rotors for bioactive polymers probing

Polysaccharides are interesting and often essential macromolecules but are difficult to analyse due to their lack of convenient chromophores. We propose an efficient labelling procedure for polysaccharides such as functionalized dextrans with coumarin derivatives: the fluorescent tracers present inter alia properties of emission of fluorescence dependent on the molecular environment (polarity, viscosity, temperature, pH, etc.). Hence, with in mind the understanding of cell-polysaccharide interactions, the labelled polymers were studied by in vitro tests on a line of endothelial cells sensitive to the proliferative effect of these dextran polysaccharides. Using 3D fluorescence microscopy, the fixation and internalization of fluorescent functionalized dextrans were observed in endothelial cells.     

Detection of liposome membrane viscosity perturbations with ratiometric molecular rotors

Molecular rotors are a form of fluorescent intramolecular charge-transfer complexes that can undergo intramolecular twisting motion upon photoexcitation. Twisted-state formation leads to non-radiative relaxation that competes with fluorescence emission. In bulk solutions, these molecules exhibit a viscosity-dependent quantum yield. On the molecular scale, the fluorescence emission is a function of the local free volume, which in turn is related to the local micro-viscosity. Membrane viscosity, and the inverse; fluidity, are characteristic terms used to describe the ease of movement withing the membrane. Often, changes in membrane viscosity govern intracellular processes and are indicative of a disease state. Molecular rotors have been used to investigate viscosity changes in liposomes and cells, but accuracy is affected by local concentration gradients and sample optical properties. We have developed self-calibrating ratiometric molecular rotors to overcome this challenge and integrated the new molecules into a DLPC liposome model exposed to the membrane-fluidizing agent propanol. We show that the ratiometric emission intensity linearly decreases with the propanol exposure and that the ratiometric intensity is widely independent of the total liposome concentration. Conversely, dye concentration inside liposomes influences the sensitivity of the system. We suggest that the new self-calibrating dyes can be used for real-time viscosity sensing in liposome systems with the advantages of lifetime measurements, but with low-cost steady-state instrumentation.     

A high-throughput approach to promoter study using green fluorescent protein

Green fluorescent protein (GFP) is a reporter that has had a significant impact due to its many advantages over other reporter genes: it is autofluorescent, it enables in situ detection, it is relatively small, and it is also nontoxic. By cloning a gene promoter upstream of the gfp gene and exposing the living cells transformed with the fusion to the specific inducer or repressor, gene expression can be real-time monitored by continuous quantitative measurement of the green fluorescence emitted by GFP. In this work, a promoter study using promoter-gfp fusions was conducted in 96-well plates. Because they were placed in an automated incubating and shaking microplate reader, the wells functioned as microscale bioreactors, allowing for parallel experiments and data analysis. In the study described here, an overexpression promoter (pBAD promoter) and two comparatively weak promoters (sodA and acnA in Escherichia coli SoxRS regulon) were studied in both endpoint and kinetics formats. Our results with the pBAD promoter revealed insight on its regulation, which is tightly controlled by levels of arabinose and glucose. Results on weak oxidative stress promoters (for sodA and acnA genes) were striking in that significant induction was observed when they were under a superoxide stress in plates. They both displayed dose-dependent induction to paraquat-generated superoxide anion, with sodA leading acnA in strength and time. These results, spanning highly inducible promoters for protein overexpression and weakly inducible promoters of metabolic interest, demonstrate that the approach is relatively easily executed and can be used for quantitative and temporal promoter studies in a high throughput format.     

A novel viscosity-sensitive fluorescent probe for monitoring the changes of mitochondrial viscosity

As a fundamental physical parameter, viscosity influences the diffusion in biological processes. The changes in intracellular viscosity led to the occurrence of relevant diseases. Monitoring changes in cellular viscosity is important for distinguishing abnormal cells in cell biology and oncologic pathology. Here, we devised and synthesized a viscosity-sensitive fluorescent probe LBX-1 . LBX-1 showed high sensitivity, providing a large Stokes shift as well as an enhancement in fluorescent intensity (16.1-fold) from methanol solution to glycerol solution. Furthermore, the probe LBX-1 could localize in mitochondria because of the ability of the probe to penetrate the cell membrane and accumulate in mitochondria. These results suggested that the probe could be utilized in monitoring the changes in mitochondrial viscosity in complex biological systems.     

Cirripede phylogeny using a novel approach: molecular morphometrics

We present a new method using nucleic acid secondary structure to assess phylogenetic relationships among species. In this method, which we term "molecular morphometrics," the measurable structural parameters of the molecules (geometrical features, bond energies, base composition, etc.) are used as specific characters to construct a phylogenetic tree. This method relies both on traditional morphological comparison and on molecular sequence comparison. Applied to the phylogenetic analysis of Cirripedia, molecular morphometrics supports the most recent morphological analyses arguing for the monophyly of Cirripedia sensu stricto (Thoracica + Rhizocephala + Acrothoracica). As a proof, a classical multiple alignment was also performed, either using or not using the structural information to realign the sequence segments considered in the molecular morphometrics analysis. These methods yielded the same tree topology as the direct use of structural characters as a phylogenetic signal. By taking into account the secondary structure of nucleic acids, the new method allows investigators to use the regions in which multiple alignments are barely reliable because of a large number of insertions and deletions. It thus appears to be complementary to classical primary sequence analysis in phylogenetic studies.     

A highly sensitive fluorescent viscosity sensor

Variation at the 3' position of fluorescein via Suzuki-Miyaura cross-coupling with aryl and heteroaryl moieties gave a family of anthofluoresceins whose spectroscopic properties were studied. The 1-methylindole derivative gave the highest quantum yield and was observed to behave as a molecular rotor, displaying marked variations in fluorescent intensities with viscosity and offering possible application in cellular sensing and fluorescent polarisation assays.     

A new approach to studies of cell-antibody interactions using fluorescent lipid probes

The interaction of poly- and monoclonal antibodies against the L-chain of human Ig with Burkitt lymphoma EB-3 cells was studied using a fluorescent lipid probe, anthrylvinyl-labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell. The possibility of using cells or liposomes carrying antigens and fluorescent lipids for the determination of antibodies in solution is discussed.     

A novel approach to estimate trabecular bone anisotropy using a database approach

Continuum finite element (FE) models of bones have become a standard pre-clinical tool to estimate bone strength. These models are usually based on clinical CT scans and material properties assigned are chosen as isotropic based only on the density distribution. It has been shown, however, that trabecular bone elastic behavior is best described as orthotropic. Unfortunately, the use of orthotropic models in FE analysis derived from CT scans is hampered by the fact that the measurement of a trabecular orientation (fabric) is not possible from clinical CT images due to the low resolution of such images. In this study, we explore the concept of using a database (DB) of high-resolution bone models to derive the fabric information that is missing in clinical images. The goal of this study was to investigate if models with fabric derived from a relatively small database can already produce more accurate results than isotropic models.     

A high resolution PIXE measurement for blood plasma ultrafiltrate

A new technique for investigation of elemental concentrations in subfractions of blood plasma is presented. The method is composed of the ultrafiltration of plasma in the presence of ethylenediaminetetraacetic acid (EDTA) and the measurement of the elemental composition by proton-induced X-ray emission (PIXE). The blood samples were collected from both healthy persons and patients suffering from breast cancer. The main emphasis in this study was on the determination of loosely bound copper (Cu) in plasma subfractions containing substances with molecular mass under 10,000, but zinc (Zn) and iron (Fe) contents of these fractions were also determined. The detection limits obtained with this method for Cu, Zn and Fe were ≈10 ppb (wet wt).