Spectroscopic characterization of the interactions between poly(2-(trimethylamino)ethyl methacrylate) chloride and the xanthene dyes, 2', 7'-difluorofluorescein and 2, 4, 5, 7-tetraiodofluorescein.

Intermolecular interactions in buffered aqueous solution between the polycation, poly(2-(trimethylamino)ethyl methacrylate) chloride (pTMAEMC) and two anionic xanthene dyes, 2', 7'-difluorofluorescein (Oregon Green 488) and 2, 4, 5, 7-tetraiodofluorescein (Erythrosin B), are characterized using multiple optical spectroscopic methods. Visible absorption spectroscopy indicates the formation of ground-state pTMAEMC-dye complexes. Benesi-Hildebrand binding isotherm analysis of visible absorption spectra for pTMAEMC-dye mixtures quantifies the strength of binding interactions producing the complexes. For both Oregon Green 488 (OG) and Erythrosin B (EB) in mixtures with pTMAEMC, the concentration of the solution's sodium acetate buffer at a fixed pH alters the binding constants, Kb, suggesting that ionic strength plays a key role in determining the binding affinity of pTMAEMC for the dyes. Comparison of Kb, for the dyes indicates stronger binding of EB under all solution conditions. Steady-state fluorescence emission spectroscopy, fluorescence quenching, excited-state fluorescence lifetime measurements and fluorescence correlation spectroscopy provide complementary data for the interactions between pTMAEMC and the dyes. Mixtures of pTMAEMC with the dyes produce fluorescence enhancements and fluorescence quenching which exhibit a dependence on the buffer concentration used in the mixture. Excited-state lifetime analysis indicates that OG interacts with pTMAEMC through ground-state interactions while EB exhibits both ground-state and excited-state interactions with pTMAEMC. The spectroscopic measurements suggest that a polyelectrolyte effect for pTMAEMC due to ionic strength variation produced by the buffer concentration affects the dye binding profile of the polycation. This conclusion is supported by fluorescence correlation spectroscopy (FCS) analyses of the hydrodynamic diameter changes in pTMAEMC-OG binding in low buffer concentration (low ionic strength) solution. FCS analyses of pTMAEMC-OG mixtures also reveal diversity in the complexes formed in low ionic strength solution suggesting that other xanthene dyes will exhibit similar binding behaviors in mixtures with pTMAEMC as a function of solution ionic strength.

Avoiding the Spherical Particle Assumption: Fractal Particle Density, Size, and Structure Characterization through Combined Sedimentation and Viscometry Measurements.

Physically meaningful characterization of irregularly shaped particles continues to present substantial challenges to the experimentalist. "Equivalent diameters" based on experimental techniques such as static and dynamic light scattering or sedimentation have proliferated to the point that they are often no longer recognized as equivalent. This study demonstrates the use of dual-fluid disk centrifuge photosedimentometry coupled with rheological measurements of viscosity to provide direct insights into both the average mass of a structured particle size distribution and the average hydrodynamic diameter.

Oligodots: Structurally Defined Fluorescent Nanoprobes for Multiscale Dual-Color Imaging in Vitro and in Vivo.

Nanoscale fluorescent probes are of great importance due to their capabilities for imaging on multiscale. Herein, we report the first synthesis of structurally well-defined nanoparticulate "oligodots" developed for multicolor imaging in vitro and in vivo. These nanoparticles are prepared via condensation and curing reactions where the engineering of the solvent results in the nanoparticles with green (λem = 550 nm) and red (λem = 650 nm) emission range. Differences found in the photophysical properties have been attributed to variations in oligomeric compositions produced during the synthesis as was corroborated by extensive physicochemical characterizations. Specifically, mass spectroscopy provided a picture of the formed species during the synthesis. The feasibility of the oligodots for multicolor imaging is demonstrated both in vitro and in vivo. The red-emitting oligodot is employed for dynamic whole-body imaging in mice. It is envisioned that oligodots would enable multicolor imaging of various biomarkers in complex diseases such as cancer where numerous molecular and metabolic phenotypes work in concert in their emergence.

Nitrogen direct analysis in real time time-of-flight mass spectrometry (N2 DART-TOFMS) for rapid screening of forensic drugs.

RATIONALE: Over the last ten years, helium direct analysis in real time time-of-flight mass spectrometry (He DART-TOFMS) has become an established technique in rapid screening of forensic drugs to decrease the time necessary to triage forensic drug cases, therefore contributing to backlog reduction and more timely criminal prosecution. Recently, we demonstrated that N2 DART was able to efficiently ionize all polar compounds except for a few extremely small ones such as methanol and acetonitrile. Therefore, N2 DART-TOFMS should be a suitable technique for rapid screening of forensic drugs. METHODS: Nitrogen direct analysis in real time time-of-flight mass spectrometry (N2 DART-TOFMS) was performed using a JEOL AccuTOF mass spectrometer with an IonSense DART-100 ion source. A 3-min analytical protocol was used for the analysis of each sample. Sample introduction was accomplished by moving the closed end of a melting point capillary where approximately 1 µL sample solution was deposited or the exposed inside of a freshly cut tablet across the N2 gas stream between the DART-100 ion source and orifice 1 of the AccuTOF. RESULTS: Ten commonly abused drugs, eight synthetic cannabinoids and four controlled prescription drugs (CPDs) were analyzed. The limit of detection (LOD) was determined to be approximately 10 µg/mL or 10 pg in quantities. All drugs at the LOD level were positively identified using their [M + H]+ ions with mass errors less than 5 mDa. The identification were further supported by in-source fragment ions and characteristic N2 DART ions that are not commonly generated by He DART, e.g. [M + H + O]+ and [M + H + 2O]+ ions. CONCLUSIONS: It was concluded that the 3-min analytical protocol could be utilized in the analysis of seized drugs in the form of tablets and powders or prepared in solution. In consideration that N2 is readily available in the air and He is a non-renewable resource, N2 DART-TOFMS is a greener, cheaper and more convenient alternative to He DART-TOFMS in rapid screening of forensic drugs.

Ionization Mechanism of Positive-Ion Nitrogen Direct Analysis in Real Time.

Nitrogen can be an inexpensive alternative to helium used by direct analysis in real time (DART), especially in consideration of the looming helium shortage. Therefore, the ionization mechanism of positive-ion N2 DART has been systematically investigated. Our experiments suggest that a range of metastable nitrogen species with a variety of internal energies existed and all of them were less energetic than metastable helium atoms. However, compounds with ionization energies (IE) equal to or lower than 10.2 eV (all organic compounds except the extremely small ones) can be efficiently ionized. Because N2 DART was unable to efficiently ionize ambient moisture and common organic solvents such as methanol and acetonitrile, the most important ionization mechanism was direct Penning ionization followed by self-protonation of polar compounds generating [M+H]+ ions. On the other hand, N2 DART was able to efficiently ionize ammonia, which was beneficial in the ionization of hydrogen-bonding compounds with proton affinities (PA) weaker than ammonia generating [M+NH4]+ ions and large PAHs generating [M+H]+ ions through proton transfer. N2 DART was also able to efficiently ionize NO, which led to the ionization of nonpolar compounds such as alkanes and small aromatics generating [M-(2m+1)H]+ (m=0,1…) ions. Lastly, metastable nitrogen species was also able to produce oxygen atoms, which resulted in increased oxygen adducts as the polarity of organic compounds decreased. In comparison with He DART, N2 DART was approximately one order of magnitude less sensitive in generating [M+H]+ ions, but could be more sensitive in generating [M+NH4]+ ions. Graphical Abstract ᅟ.

Infrared Spectroscopic Analysis of the Adsorption of Pyridine Carboxylic Acids on Colloidal Ceria.

Surface adsorption of a homologous series of pyridine carboxylic acids on a hydrated colloidal cerium dioxide (ceria) film is characterized using the combination of experimental and computationally determined infrared (IR) spectra. Experimental analyses employ attenuated total reflectance (ATR) IR spectroscopy of deposited colloidal ceria thin films equilibrated with three pyridine carboxylic acids at pH 3.0, 5.5, and 8.5. The corresponding computational IR spectra for the energy-minimized intermediate and base forms of the pyridine carboxylic acids use density functional theory calculations at the B3LYP/6-311++G** level of theory. Solvent effects are modeled using both the COSMO implicit solvation model and the inclusion of explicit water molecules. Experimental IR spectra show that the adsorptive interactions between the pyridine carboxylic acids and ceria surface are due to the outer-sphere coordination of cerium ions in the films. Vibrational assignments based on combined experimental and computational results indicate that both pyridyl ring nitrogen and carboxylate functional groups account for the interaction of pyridine carboxylic acids at ceria surfaces. Experimentally determined Langmuir constants point to the intermediate form of picolinic acid (pyridine-2-carboxylic acid) as having the strongest adsorption to ceria compared to the other pyridine carboxylic acids investigated. The enhanced adsorption of picolinic acid is attributed to the adjacency of the protonated pyridyl nitrogen and the carboxylate group relative to nicotinic acid (pyridine-3-carboxylic acid) and isonicotinic acid (pyridine-4-carboxylic acid).

Reduction of artifacts in fluorescence correlation spectroscopy due to sample adsorption on optical glass surfaces.

The preparation of glass cell surfaces that are chemically functionalized with poly(ethylene glycol) (PEG) chains to reduce sample adsorption and their use in fluorescence correlation spectroscopy (FCS) is described. Optical glass coverslips were acid etched and reacted with either 750 Mr PEG (PEG-750) or 5000 Mr PEG (PEG-5000) to produce adsorption-resistant optical surfaces. FCS data for Nile red-loaded Triton X-100 micelles (NR-TX-100) and Alexa Fluor 555-labeled proteins, bovine serum albumin (BSA-A555), lipidized BSA (lipid-BSA-A555), and three low molecular weight dyes deposited on PEGylated coverslips were evaluated. Measurement artifacts due to sample adsorption on the PEG-5000 functionalized coverslips were reduced significantly for the majority of test materials. Calculations of translational diffusion coefficients and Stokes radii confirmed the effectiveness of this approach. PEG-5000 functionalized coverslips were demonstrated as more effective in inhibiting adsorption than PEG-750 functionalized coverslips. Neither of the functionalized coverslips inhibited the adsorption of one test compound, rhodamine B, a dye that adsorbs strongly on glass surfaces. The use of longer PEG chains in conjunction with chemical cross-linking is proposed for producing a denser, less porous PEG layer for the prevention of strongly glass-adsorbing fluorophores that do not interact with the PEG layer.

Simultaneous absolute determination of particle size and effective density of submicron colloids by disc centrifuge photosedimentometry.

Disc centrifuge photosedimentometry (DCP) with fluids of different densities is used to simultaneously determine the particle size and effective density of spherical silica particles. Incorporation of a calibrated infrared pyrometer into a DCP instrument is shown to enhance the measurement capability of the DCP technique by correcting for the temperature dependence of the spin fluid's density and viscosity. Advantages of absolute DCP determinations for size and density analysis relative to standardized DCP measurements include the elimination of instrument standardization with a particle of known density and measurements or estimation of the effective particle density. The reliability of diameter determinations provided by absolute DCP was confirmed using silica particles with nominal diameters ranging from 250 to 700 nm by comparison of these analyses with a diameter determination by transmission electron microscopy for silica particle size standards. Effective densities determined by absolute DCP for the silica particles ranged from 2.02 to 2.34 g/cm(3). These findings indicate that the silica particles have little or no porosity. The reported characterization of colloidal silica using absolute DCP suggests applicability of the technique to a variety of particle types including colloidal materials other than silica, core-shell particles, compositionally heterogeneous mixtures of nanoparticles, and irregularly shaped, structured colloids.

Application of SIMSTEX to oligomerization of insulin analogs and mutants.

The propensity of various insulins and their analogs to oligomerize was investigated by mass spectrometric methods including measurement of the relative abundances of oligomers in the gas phase and the kinetics of H/D amide exchange. The kinetics of deuterium uptake show a good fit when the exchanging amides are placed in three kinetic groups: fast, intermediate, and slow. r-Human insulin, of the insulins investigated, has fewer amides that exchange at intermediate rates and more that exchange at slow rates, in accord with its higher extent of association in solution. We adapted PLIMSTEX (protein ligand interactions by mass spectrometry, titration, and H/D exchange) to determine protein/ligand affinities in solution, to determine self-association equilibrium constants for proteins, and to apply them to various insulin analogs. We term this adaptation SIMSTEX (self-association interactions using mass spectrometry, self-titration and H/D exchange); it gives affinity constants that compare well with the literature results. The results from SIMSTEX show that some mutants (e.g., GlnB13) have an increased tendency to self-associate, possibly slowing down their action in vivo. Other mutants (e.g., lispro and AspB9) have lower propensities for self-association, thus providing potentially faster-acting analogs for use in controlling diabetes.

The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins.

The carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad-spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel-chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium-throughput HPLC assay using a fluorescent substrate and a high-throughput assay based on fluorescence polarization capable of application in a high-throughput 96-well plate format. This high-throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their K(m) and V(max) values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size-exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed.

Peptide-derivatized shell-cross-linked nanoparticles. 1. Synthesis and characterization.

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell-cross-linked (SCK) nanoparticles is reported as a method to facilitate cell surface binding and transduction of SCK nanoparticles. Attaching increasing numbers of peptide sequences to SCK nanoparticles in a global solution-state functionalization strategy has been devised as a method for increasing the efficiency of the cell-penetrating process. The numbers of peptides per SCK were controlled through stoichiometric balance and measured experimentally by two independent methods, UV-visible spectroscopy and phenylglyoxal analysis. PTD was conjugated in (0.005, 0.01, and 0.02) molar ratios, relative to the acrylic acid residues in the shell, to the SCK nanoparticles resulting in SCK populations possessing nominally 52, 104, and 210 (41, 83, and 202 as measured by phenylglyoxal analysis) PTD peptides per particle, respectively. The methodologies for the block copolymer and nanoparticle syntheses, peptide derivatization, and characterization of peptide-functionalized SCK nanoparticles are reported and the feasibility and efficiency of intracellular internalization of the respective SCKs were quantified.

Synthesis, characterization, and bioavailability of mannosylated shell cross-linked nanoparticles.

Saccharide-functionalized shell cross-linked (SCK) polymer micelles designed as polyvalent nanoscaffolds for selective interactions with receptors on Gram negative bacteria were constructed from mixed micelles composed of poly(acrylic acid-b-methyl acrylate) and mannosylated poly(acrylic acid-b-methyl acrylate). The mannose unit was conjugated to the hydrophilic chain terminus of the amphiphilic diblock copolymer precursor, from which the SCK nanoparticles were derived, by the growth of the diblock copolymer from a mannoside functionalized atom transfer radical polymerization (ATRP) initiator. Mixed micelle formation between the amphiphilic diblock copolymer and mannosylated amphiphilic diblock copolymer was followed by condensation-based cross-linking between the acrylic acid residues present in the periphery of the polymer micelles to afford SCK nanoparticles. SCKs presenting variable numbers of mannose functionalities were prepared from mixed micelles of controlled stoichiometric ratios of mannosylated and nonmannosylated diblock copolymers. The polymer micelles and SCKs were characterized by dynamic light scattering (DLS), electrophoretic light scattering, atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). Surface availability and bioactivity of the mannose units were evaluated by interactions of the nanostructures with the model lectin Concanavalin A via DLS studies, with red blood cells (rabbit) via agglutination inhibition assays and with bacterial cells (E. coli) via TEM imaging.

Determination of the bioavailability of biotin conjugated onto shell cross-linked (SCK) nanoparticles.

Shell cross-linked nanoparticles (SCKs) presenting surface- and bioavailable biotin functional groups were synthesized via a mixed micelle methodology, whereby co-micellization of chain terminal biotinylated poly(acrylic acid)-b-poly(methyl acrylate) (PAA-b-PMA) and nonbiotinylated PAA-b-PMA were cross-linked in an intramicellar fashion within the shell layer of the mixed micelles, between the carboxylic acid groups of PAA and the amine functionalities of 2,2'-(ethylenedioxy)diethylamine. The hydrodynamic diameters (D(h)) of the micelles and the SCKs with different biotinylated block copolymer contents were determined by dynamic light scattering (DLS), and the dimensions of the SCKs were characterized with tapping-mode atomic force microscopy (AFM) and transmission electron microscopy (TEM). The amount of surface-available biotin was tuned by varying the stoichiometric ratio of the biotinylated PAA-b-PMA versus the nonbiotinylated PAA-b-PMA, as demonstrated with solution-state, binding interaction analyses, an avidin/HABA (avidin/4'-hydroxyazobenzene-2-carboxylic acid) competitive binding assay, and fluorescence correlation spectroscopy (FCS). The avidin/HABA assay found the amount of available biotin at the surface of the biotinylated SCK nanoparticles to increase with increasing biotin-terminated block copolymer incorporation, but to be less than 25% of the theoretical value. FCS measurements showed the same trend.

Chemically induced supramolecular reorganization of triblock copolymer assemblies: trapping of intermediate states via a shell-crosslinking methodology.

The mechanism of morphological phase transitions was studied for rod-shaped supramolecular assemblies comprised of a poly(acrylic acid)-block-poly(methyl acrylate)-block-polystyrene (PAA(90)-b-PMA(80)-b-PS(100)) triblock copolymer in 33% tetrahydrofuran/water after perturbation by reaction with a positively charged water-soluble carbodiimide. Tetrahydrofuran solvation of the hydrophobic core domain provided the dynamic nature required for the rod-to-sphere phase transition to be complete within 30 min. The intermediate morphologies such as fragmenting rods and pearl-necklace structures were trapped kinetically by the subsequent addition of a diamino crosslinking agent, which underwent covalent crosslinking of the shell layer. Alternatively, shell-crosslinked rod-shaped nanostructures with preserved morphology were obtained by the addition of the crosslinking agent before the addition of the carbodiimide, which allowed for the shell crosslinking to be performed at a faster rate than the morphological reorganization. The formation of robust shell-crosslinked nanostructures provides a methodology by which the morphological evolution processes can be observed, and it allows access to otherwise thermodynamically unstable nanostructures.