Charged Fluorophores Research Articles

Accelerated Activity-Based Sensing by Fluorogenic Reporter Engineering Enables to Rapidly Determine Unstable Analyte.

Accurate detection of labile analytes through activity based fluorogenic sensing is meaningful but remains a challenge because of nonrapid reaction kinetic. Herein, we present a signaling reporter engineering strategy to accelerate azoreduction reaction by positively charged fluorophore promoted unstable anion recognition for rapidly sensing sodium dithionite (Na2S2O4), a kind of widespread used but harmful inorganic reducing agent. Its quick decomposition often impedes application reliability of traditional fluorogenic probes in real samples because of their slow responses. In this work, four azo-based probes with different charged fluorophores (positive, zwitterionic, neutral, and negative) were synthesized and compared. Among of them, with sequestration effect of positively charged anthocyanin fluorophore for dithionite anion via electrostatic attraction, the cationic probe Azo-Pos displayed ultrafast fluorogenic response (∼2 s) with the fastest response kinetic (kpos' = 0.373 s-1) that is better than other charged ones (kzwi' = 0.031 s-1, kneu' = 0.013 s-1, kneg' = 0.003 s-1). Azo-Pos was demonstrated to be capable to directly detect labile Na2S2O4 in food samples and visualize the presence of Na2S2O4 in living systems in a timely fashion. This new probe has potential as a robust tool to fluorescently monitor excessive food additives and biological invasion of harmful Na2S2O4. Moreover, our proposed accelerating strategy would be versatile to develop more activity-based sensing probes for quickly detecting other unstable analytes of interest.

Self-Quenching Behavior of a Fluorescent Probe Incorporated within Lipid Membranes Explored Using Electrophoresis and Fluorescence Lifetime Imaging Microscopy.

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility, and interactions of biomolecules. However, fluorophores can undergo "self-quenching" of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artifacts in fluorescence images and relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores associated with supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of lipid-linked Texas Red (TR) fluorophores were generated within 100 × 100 μm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively charged TR-lipid molecules toward the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mol/mol), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mol/mol), leading to the reduction of fluorescence lifetime down to 30% and quenching of the fluorescence intensity down to 10% of their original levels. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The calculated concentration profiles have a good fit to an exponential growth function, suggesting that TR-lipids can diffuse freely even at high concentrations. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

Exploring the self-quenching of fluorescent probes incorporated into model lipid membranes using electrophoresis and fluorescence lifetime imaging microscopy.

Fluorescent probes are useful in biophysics research to assess the spatial distribution, mobility and interactions of biomolecules. However, fluorophores can undergo “self-quenching” of their fluorescence intensity at high concentrations. A greater understanding of concentration-quenching effects is important for avoiding artefacts in fluorescence images and is relevant to energy transfer processes in photosynthesis. Here, we show that an electrophoresis technique can be used to control the migration of charged fluorophores within supported lipid bilayers (SLBs) and that quenching effects can be quantified with fluorescence lifetime imaging microscopy (FLIM). Confined SLBs containing controlled quantities of Texas Red (TR) fluorophores were generated within 100 × 100 µm corral regions on glass substrates. Application of an electric field in-plane with the lipid bilayer induced the migration of negatively-charged TR molecules towards the positive electrode and created a lateral concentration gradient across each corral. The self-quenching of TR was directly observed in FLIM images as a correlation of high concentrations of fluorophores to reductions in their fluorescence lifetime. By varying the initial concentration of TR fluorophores incorporated into the SLBs from 0.3% to 0.8% (mole/mole), the maximum concentration of fluorophores reached during electrophoresis could be modulated from 2% up to 7% (mole/mole), leading to maximal quenching of 30% up to 70%. As part of this work, we demonstrated a method for converting fluorescence intensity profiles into molecular concentration profiles by correcting for quenching effects. The shape of the calculated concentration profiles revealed evidence of subtle lipid-lipid interactions at high packing densities. Overall, these findings prove that electrophoresis is effective at producing microscale concentration gradients of a molecule-of-interest and that FLIM is an excellent approach to interrogate dynamic changes to molecular interactions via their photophysical state.

Intramolecular photo-driven charge transfer in a series of pyridyl substituted phenyloxazoles. Structural relaxation in meta-substituted ethylpyridinium derivative of phenyloxazole.

A series of pyridyl (pyridinium) substituted benzoxazoles were studied by steady state absorption, fluorescence spectroscopy, time-resolved fluorescence spectroscopy, fs pulse absorption and polarization spectroscopy, and quantum-chemical calculations. The spectral and kinetic parameters of the fluorophores in MeCN and EtOAc were obtained experimentally and were calculated by means of DFT and TDDFT methods. A scheme including four transient excited states was proposed for the interpretation of differential absorption kinetics of the charged fluorophores. Expressions describing the actual kinetics graphs, the decay associated spectra, and the species-associated spectra were derived. The charge shift step was found to be dependent on average solvation times. A charge shift followed by the formation of the twisted conformer was found for the excited 1-ethyl-3-(5-phenyloxazol-2-yl)pyridinium 4-methyl-1-benzenesulfonate in MeCN and EtOAc. Conformational analysis confirms a large amplitude motion of the meta-substituted ethylpyridinium group as an additional structural relaxation path producing an abnormally large fluorescence Stokes shift.

Pitfalls in Monitoring Mitochondrial Temperature Using Charged Thermosensitive Fluorophores

Mitochondria are the source of internal heat which influences all cellular processes. Hence, monitoring mitochondrial temperature provides a unique insight into cell physiology. Using a thermosensitive fluorescent probe MitoThermo Yellow (MTY), we have shown recently that mitochondria within human cells are maintained at close to 50 °C when active, increasing their temperature locally by about 10 °C. Initially reported in the HEK293 cell line, we confirmed this finding in the HeLa cell line. Delving deeper, using MTY and MTX (MitoThermo X), a modified version of MTY, we unraveled some caveats related to the nature of these charged fluorophores. While enabling the assessment of mitochondrial temperature in HEK and HeLa cell lines, the reactivity of MTY to membrane potential variations in human primary skin fibroblasts precluded local temperature monitoring in these cells. Chemical modification of MTY into MTX did not result in a temperature probe unresponsive to membrane potential variations that could be universally used in any cell type to determine mitochondrial temperature. Thus, the cell-type dependence of MTY in measuring mitochondrial temperature, which is likely due to the variable binding of this dye to specific internal mitochondrial components, should imply cautiousness while using these nanothermometers for mitochondrial temperature analysis.

To What Extent Do Fluorophores Bias the Biological Activity of Peptides? A Practical Approach Using Membrane-Active Peptides as Models.

The characterization of biologically active peptides relies heavily on the study of their efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location, using both in vitro and in vivo studies. These studies frequently depend on the use of fluorescence-based techniques. Since most peptides are not intrinsically fluorescent, they are conjugated to a fluorophore. The conjugation may interfere with peptide properties, thus biasing the results. The selection of the most suitable fluorophore is highly relevant. Here, a comprehensive study with blood–brain barrier (BBB) peptide shuttles (PepH3 and PepNeg) and antimicrobial peptides (AMPs) (vCPP2319 and Ctn[15-34]), tested as anticancer peptides (ACPs), having different fluorophores, namely 5(6)-carboxyfluorescein (CF), rhodamine B (RhB), quasar 570 (Q570), or tide fluor 3 (TF3) attached is presented. The goal is the evaluation of the impact of the selected fluorophores on peptide performance, applying routinely used techniques to assess cytotoxicity/toxicity, secondary structure, BBB translocation, and cellular internalization. Our results show that some fluorophores significantly modulate peptide activity when compared with unlabeled peptides, being more noticeable in hydrophobic and charged fluorophores. This study highlights the need for a careful experimental design for fluorescently labeled molecules, such as peptides.

Discharging behavior of confined bipolar electrodes: Coupled electrokinetic and electrochemical dynamics

In this study we numerically investigate the intimately coupled transient electrokinetic and electrochemical dynamics of a nanoconfined bipolar electrode system, using experimental results to guide our analysis. Our recently developed 2D numerical model implements the Poisson-Nernst-Planck-Stokes system of equations to describe nanoscale chemical species transport by advection, migration, and diffusion, as well as the presence of both homogenous and heterogeneous reactions. By eschewing the assumption of electroneutrality and resolving diffuse-charge screening effects, our model uniquely accounts for a wide range of nonlinear transient effects including bipolar electrode (BPE) surface polarization, Faradaic charge accumulation, induced-charge electroosmotic flow, and ion concentration polarization. Using this model, we demonstrate that upon the removal of a polarizing electric field, excess accumulated charge at a BPE surface electrochemically discharges following the capacitive relaxation of diffuse space charge in the electrical double layers (EDLs) surrounding the BPE extremities. These EDLs continue to polarize the BPE as they relax by a process of drift-diffusion, wherein the counter-ionic space charge at each pole promotes a large influx of oppositely charged ions to restore electroneutrality. We numerically reproduced this electrokinetic enhancement effect that was first observed in a recently reported experimental system in which charged fluorophores were used as tracer molecules. Our results also support experimental evidence that, following capacitive EDL relaxation, nanoconfined BPEs can exhibit pseudocapacitance-like discharging behavior that is localized to a single end of the electrode; we experimentally linked this localization to surface oxidation of the anodic BPE pole under the applied field. In addition to providing important insight into the interplay between nanoscale electrokinetic and electrochemical phenomena that govern transient electrode processes, our model and the results presented in this work reinforce the notion that the domain of bipolar electrochemistry constitutes a promising frontier for developing “wirelessly” tunable charge storage and visual detection approaches which exploit both electrokinetic and Faradaic mechanisms.

Fluorescence-Based Observation of Transient Electrochemical and Electrokinetic Effects at Nanoconfined Bipolar Electrodes.

Bipolar electrodes (BPEs) are conductors that, when exposed to an electric field, polarize and promote the accumulation of counterionic charge near their poles. The rich physics of electrokinetic behavior near BPEs has not yet been rigorously studied, with our current understanding of such bipolar effects being restricted to steady-state conditions (under constant applied fields). Here, we reveal the dynamic electrokinetic and electrochemical phenomena that occur near nanoconfined BPEs throughout all stages of a reaction. Specifically, we demonstrate, both experimentally and through numerical modeling, that the removal of an electric field produces solution-phase charge imbalances in the vicinity of the BPE poles. These imbalances induce intense and short-lived nonequilibrium electric fields that drive the rapid transport of ions toward specific BPE locations. To determine the origin of these electrokinetic effects, we monitored the movement and fluorescent behavior (enhancement or quenching) of charged fluorophores within well-defined nanofluidic architectures via real-time optical detection. By systematically varying the nature of the fluorophore, the concentration of the electrolyte, the strength of the applied field, and oxide growth on the BPE surface, we dissect the ion transport events that occur in the aftermath of field-induced polarization. The results contained in this work provide new insights into transient bipolar electrokinetics that improve our understanding of current analytical platforms and can drive the development of new micro- and nanoelectrochemical systems.

Ion concentration in micro and nanoscale electrospray emitters.

Solution-phase ion transport during electrospray has been characterized for nanopipettes, or glass capillaries pulled to nanoscale tip dimensions, and micron-sized electrospray ionization emitters. Direct visualization of charged fluorophores during the electrospray process is used to evaluate impacts of emitter size, ionic strength, analyte size, and pressure-driven flow on heterogeneous ion transport during electrospray. Mass spectrometric measurements of positively- and negatively-charged proteins were taken for micron-sized and nanopipette emitters under low ionic strength conditions to further illustrate a discrepancy in solution-driven transport of charged analytes. A fundamental understanding of analyte electromigration during electrospray, which is not always considered, is expected to provide control over selective analyte depletion and enrichment, and can be harnessed for sample cleanup. Graphical abstract Fluorescence micrographs of ion migration in nanoscale pipettes while solution is electrosprayed.

DUTPs conjugated with zwitterionic Cy3 or Cy5 fluorophore analogues are effective substrates for DNA amplification and labelling by Taq polymerase.

To develop structural modifications of dNTPs that are compatible with Taq DNA polymerase activity, we synthesized eight dUTP derivatives conjugated with Cy3 or Cy5 dye analogues that differed in charge and charge distribution throughout the fluorophore. These dUTP derivatives and commercial Cy3− and Cy5-dUTP were studied in Taq polymerase-dependent polymerase chain reactions (PCRs) and in primer extension reactions using model templates containing one, two and three adjacent adenine nucleotides. The relative amounts of amplified DNA and the kinetic parameters Km and Vmax characterizing the incorporation of labelled dUMPs have been estimated using fluorescence measurements and analysed. The dUTPs labelled with electroneutral zwitterionic analogues of Cy3 or Cy5 fluorophores were used by Taq polymerase approximately one order of magnitude more effectively than the dUTPs labelled with negatively charged analogues of Cy3 or Cy5. The nucleotidyl transferase activity of Taq polymerase was also observed and resulted in the addition of dUMPs labelled with electroneutral or positively charged fluorophores to the 3′ ends of DNA. The introduction of mutually compensating charges into fluorophores or other functional groups conjugated to dNTPs can be considered a basis for the creation of PCR-compatible modified nucleoside triphosphates.

Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical.

Lanthanide-based metal-organic framework nanosheets with unique fluorescence quenching properties for two-color intracellular adenosine imaging in living cells

Two-dimensional (2D) materials have attracted tremendous interest as fluorescence quenchers of dye-labeled biomolecules for application in biosensing. Metal-organic framework (MOF) nanosheets, as a new type of 2D material, have rarely been studied as bioanalytical platforms. Herein, we synthesize a series of ultrathin lanthanide-based MOF (MOF-Ln) nanosheets as a dye-labeled aptamer platform. The fluorescence quenching or recovery on the MOF-Ln nanosheets is determined by the charge properties (positive or negative) of the labeled fluorophores. The negatively charged fluorophores experience a fluorescence ‘turn-down followed by turn-down’ process, whereas the positively charged fluorophores experience a fluorescence ‘turn-down followed by turn-up’ process. The interesting fluorescence quenching properties of the MOF-Ln nanosheets make them an excellent two-color sensing platform for the intracellular detection of biomolecules.

Abstract 4305: Biodistribution and clearance of non-targeted DyLight dyes in tumor-free nude mice for in vivo imaging

Abstract Far red to near infra red (NIR) fluorophores are commonly used in cell-based assays and for deep tissue in vivo imaging. The light emitting and physico-chemical characteristics of these fluorophores, e.g. excitation and emission spectra, relative hydrophilicity/hydrophobicity and molecule size, can significantly influence their performance in these applications. Currently there are no guidelines for selection of fluorophores optimal for targeted in vivo imaging. To address this issue we have designed a panel of DyLight dyes with maximum excitation wavelength in the range of 650 -785 nm, maximum emission wavelength in the range of 670 - 800 nm and different levels of sulfonation and/or PEGylation. We studied the kinetic of tissue distribution and clearance of these fluorophores as well as their effect on morphology of different tissues in mice after intravenous injection. Briefly, nude mice were anesthetized, injected with a dye (100 µL of dye at 0.5mg/ml in PBS) into retro-orbital plexus, and imaged using Carestream MSFX or Carestream XTREME Imager. Images were generated before dye injection, immediately after injection (0 hours) and 3, 6, 12 and 24 hours post injection. At the end of the experiment, animals were sacrificed; internal organs were collected and used for ex vivo analysis. The results of in vivo imaging experiments demonstrated that: hydrophobic fluorophores have a relatively slow kinetic of distribution and clearance, and often cleared via a hepatic pathway. Introduction of the negatively charged groups and or PEG chains increased dye solubility affected the kinetics of both fluorophore distribution and its clearance. In addition, negatively charged fluorophores had a tendency to be cleared predominantly via renal pathway. A histological analysis of the internal organs showed no detectable tissue damage. These results provide guidelines for the selection of optimal NIR dyes for in vivo imaging. Citation Format: Marie C. NLEND, Surbhi Desai, Suk J. Hong, Mary Beth Anderson, Georgyi V. Los, Greg T. Hermanson, Justin M. Diener, Warren M. Leevy, Peter A. Bell. Biodistribution and clearance of non-targeted DyLight dyes in tumor-free nude mice for in vivo imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4305. doi:10.1158/1538-7445.AM2014-4305

Characterization of solute distribution following iontophoresis from a micropipet.

Iontophoresis uses a current to eject solution from the tip of a barrel formed from a pulled glass capillary and has been employed as a method of drug delivery for neurochemical investigations. Much attention has been devoted to resolving perhaps the greatest limitation of iontophoresis, the inability to determine the concentration of substances delivered by ejections. To further address this issue, we evaluate the properties of typical ejections such as barrel solution velocity and its relation to the ejection current using an amperometric and liquid chromatographic approach. These properties were used to predict the concentration distribution of ejected solute that was then confirmed by fluorescence microscopy. Additionally, incorporation of oppositely charged fluorophores into the barrel investigated the role of migration on the mass transport of an ejected species. Results indicate that location relative to the barrel tip is the primary influence on the distribution of ejected species. At short distances (<100 μm), advection from electroosmotic transport of the barrel solution may significantly contribute to the distribution, but this effect can be minimized through the use of low to moderate ejection currents. However, as the distance from the source increases (>100 μm), even solute ejected using high currents exhibits diffusion-limited behavior. Lastly a time-dependent theoretical model was constructed and is used with experimental fluorescent profiles to demonstrate how iontophoresis can generate near-uniform concentration distributions near the ejection source.

A polylysine-based fluorescent probe for sulfite anion detection in aqueous media via analyte-induced charge generation and complexation

Sulfite is widely used as a preservative in food and beverages; and human exposure to SO2 (which is easily transformed into sulfite) has become increasingly widespread. Overdose or inhalation of sulfite may induce many respiratory responses and a number of diseases. Hence, it is important to achieve rapid, sensitive and selective detection of sulfites. Herein, we report a robust fluorescent turn-on probe for sulfite anions via the analyte-induced charge generation and AIE effect (the aggregation induced emission effect). The two-component sensing system comprises a modified polylysine with benzoic aldehyde side groups and a positively charged tetraphenylethene bearing two quaternary ammonium groups. In the presence of sulfite anions, the benzoic aldehyde groups on polylysine side chains react with sulfite and turn into negatively charged groups, leading to the complexation between the polymer chains and the positively charged fluorophores (TPE-2N+) and eventually achieving the fluorescence enhancement of the system as a result of the AIE effect. The integration of the water-soluble biopolymer and AIE-active fluorophore ensures that the system is operable in totally aqueous media (100% water), which is beneficial for applications in food quality control, environmental pollution monitoring and biological assays. Furthermore the system exhibits highly selective and sensitive fluorescent turn-on detection for sulfite, with the detection limit of 3.6 μM. Moreover, it can be used in real samples such as red wine, beer and rainwater, and there is no need for special sample pretreatment.

Simple strategies towards bright polymer particles via one-step staining procedures

In order to develop simple and versatile procedures for the preparation of red emissive particles, various one-step swelling procedures for the loading of fluorophores into nanometer- and micrometer-sized polystyrene particles were systematically assessed. Parameters studied for model dyes from common dye classes include the composition of the swelling medium, dye charge and polarity, dye concentration, and particle surface chemistry. The dye loading procedures were compared based upon the efficiency of dye incorporation, fluorescence intensity, and colloidal stability of the resulting particles as well as the absence of dye leaking as determined by absorption and fluorescence spectroscopy, flow cytometry, and measurements of zeta potentials. In addition, for the first time, the influence of the amount of incorporated dye on the absolute fluorescence quantum yield and brightness of the fluorescent particles was investigated for selected chromophores in differently sized particles using a custom-made calibrated integrating sphere setup. Our results demonstrate the general suitability of these one-step loading procedures for efficient particle staining with neutral, zwitterionic, and charged fluorophores like oxazines, coumarines, squaraines, xanthenes, and cyanines emitting in the visible and near infrared. Dye polarity was identified as a suitable tool to estimate the loading efficiency of fluorophores into these polymer particles.

Optical detection of DNA hybridization based on fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles

A novel system for the detection of DNA hybridization in a homogeneous format is developed. This method is based on fluorescence quenching by gold nanoparticles used as both nanoscaffolds for the immobilization of capture sequences and nanoquenchers of fluorophores attached to detection sequences. The oligonucleotide-functionalized gold nanoparticles are synthesized by derivatizing the colloidal gold solution with 5′-thiolated 12-base oligonucleotides. Introduction of sequence-specific target DNAs (24 bases) into the mixture containing dye-tagged detection sequences and oligonucleotide-functionalized gold nanoparticles results in the quenching of carboxytetramethylrhodamine-labeled DNA fluorescence because DNA hybridization occurs and brings fluorophores into close proximity with oligonucleotide-functionalized gold nanoparticles. The quenching efficiency of fluorescence increases with the target DNA concentration and provides a quantitative measurement of sequence-specific DNA in sample. A linearity is obtained within the range from 1.4 to 92 nM. The target sequence is detected down to 2 nM. This new system not only overcomes many of the drawbacks inherent in radioisotopic measurement or enzyme-linked assay but also avoids the requirement for the stem–loop structure compared with conventional molecular beacons. Furthermore, the background signal that is defined as fluorescence quenching arising from electrostatic attraction between positively charged fluorophores and negatively charged gold nanoparticles is comparatively low due to electrostatic repulsion between negatively charged oligonucleotides. In addition, this is a homogeneous assay that can offer the potential to be monitored in real time, be amenable to automation, eliminate washing steps, and reduce the risk of contamination.

Fluorophore-assisted carbohydrate electrophoresis: a sensitive and accurate method for the direct analysis of dolichol pyrophosphate-linked oligosaccharides in cell cultures and tissues

Lipid-linked oligosaccharides (LLOs) such as Glc 3Man 9GlcNAc 2-P-P-dolichol are the precursors of asparagine (N)-linked glycans, which are essential information carriers in many biological systems, and defects in LLO synthesis cause Type I Congenital Disorders of Glycosylation. Due to the low abundance of LLOs and the limitations of the chemical and physical methods previously used to detect them, almost all studies of LLO synthesis have relied upon metabolic labeling of the oligosaccharides with radioactive sugar precursors such as [ 3H]mannose or [ 14C]glucosamine. In this article, a procedure is presented for a facile, accurate, and sensitive non-radioactive method for LLO analysis based on fluorophore-assisted carbohydrate electrophoresis (FACE). First, LLOs are extracted and partially purified. Next, oligosaccharides released from LLOs are labeled with negatively charged fluorophores: 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) or 7-amino-1,3-naphthalenedisulfonic acid (ANDS). A specialized form of polyacrylamide gel electrophoresis is then used to resolve and measure ANTS or ANDS labeled oligosaccharides. Finally, the resolved oligosaccharides are detected and quantified by fluorescence imagers using CCD cameras.

Incorporation of fluorescent molecules and proteins into calcium oxalate monohydrate single crystals

A central question to understanding biomineralization is determining how biomolecules are integrated within inorganic host lattices, thereby changing material properties yet retaining single crystal structure of the biomineral. We have addressed guest incorporation within single biomineral crystals by investigating face specificity, anisotropy and the role of charges in fluorescent calcium oxalate monohydrate (COM) crystals nucleated from solutions containing eosin Y, fluoresceins and rhodamines. Additionally, we have examined the specificity of incorporation of Protein G wild-type (G-wt) and its mutant (G-Δ6, in which four aspartic acids and two glutamic acids have been replaced by the corresponding asparagine or glutamine), where G-wt promotes and G-Δ6 inhibits COM crystal growth. We found that (1) the negatively charged fluorophores, as well as the fluorophore-labeled proteins, G-wt and G-Δ6, were successfully incorporated during growth into the same {1 1 0} growth sectors in preference to all others; (2) the positively charged TRITC (tetramethyl rhodamine isothiocyanate) was not incorporated as free fluorophore, but it became incorporated if conjugated to G-wt and G-Δ6; (3) once the fluorophores are incorporated, the polarization measurements of adsorption and emission were similar irrespectively whether taken from COM containing the free fluorophores, or the protein conjugates. The anisotropy was similar for rhodamines and fluoresceins.

Concentration-dependent fluorescence quenching and electron scavenging in liquids

We present a theory for the dependence of the fluorescence quenching lifetime τ on quencher concentration and the dependence of the electron-scavenger rate constant kf on scavenger concentration. At low quencher (scavenger) concentration these quantities are obtained by solution of the Smoluchowski–Debye diffusion equation. For nondilute quenchers we find positive deviations from the linear Stern–Volmer equation for 1/τ and for nondilute scavengers we find positive deviations from linear behavior in kf. With reactants that interact by a long range potential, such as charged fluorophores and quenchers, and electrons with charged scavengers, a length ã∼100–300 Å vs the collisional encounter length a ∼1–10 Å is the significant length. Corrections to dilute behavior are found to depend on the effective volume fraction of quenchers (or scavengers) φ̃=4π3ã3c/3 which can be much larger than the material volume fraction φ=4π3a3c/3 [c is the quencher (or scavenger) number density]. We consider both the initial value and steady state situations and find that, for our exact results, no transient behavior of the rate coefficient enters the initial value expression.