Aqueous Phosphate-buffered Saline Research Articles

Dewetting of Polymer Films Controlled by Protein Adsorption.

The stability of the film poly(n-butyl methacrylate) (PnBMA) with different tacticities, prepared on silicon oxide and exposed to aqueous phosphate-buffered saline with different concentrations of bovine serum albumin (CBSA between 0 and 4.5 mg/mL), was examined at temperatures close to the physiological limit (between 4 and 37 °C) with optical microscopy, contact angle measurements, atomic force microscopy, and time-of-flight secondary ion mass spectrometry. For PBS solutions with CBSA = 0, the stability of atactic PnBMA and dewetting of isotactic PnBMA was observed, caused by the interplay between the stabilizing long-range dispersion forces and the destabilizing short-range polar interactions. Analogous considerations of excess free energy cannot explain the retardation of dewetting observed for isotactic PnBMA in PBS solutions with higher CBSA. Instead, formation of a BSA overlayer, adsorbed preferentially but not exclusively to uncovered SiOx regions, is evidenced and postulated to hinder polymer dewetting. Polymer dewetting and protein patterning are obtained in one step, suggesting a simple approach to fabricate biomaterials with micropatterned proteins.

Chain flexibility and dynamics of alginate solutions in different solvents

Mechanical rheometry, specifically rotational rheometry, squeeze flow, and capillary rheometry, and two microrheology methods, namely multiple-particle tracking (MPT) and diffusing wave spectroscopy (DWS) have been used to get new insight into structural and dynamical properties of alginate dissolved in solvents widely used for bioprinting, namely deionized water, phosphate-buffered saline (PBS), and Dulbecco Modified Eagle Medium (DMEM) cell media. Results demonstrate that alginate rheological properties depend on the solvent quality at concentrations higher than 1 wt.%. In this high concentration regime, in aqueous salt-free and PBS solutions, experimental scaling exponents for the concentration dependence of the specific viscosity ηsp and the plateau modulus G0 agree well with theoretical predictions for neutral polymers in good solvent whereas for the terminal relaxation time TR, the exponent is slightly higher than theoretically predicted, presumably due to the formation of aggregates. For alginate dissolved in DMEM, all exponents for ηsp, G0, and TR agree with predictions for polymers in theta solvents, which might be related to the formation of polyelectrolyte complex as a result of interactions between alginate and amino acids. Chain persistence length lp values, as determined directly from high frequency rheometry for the first time, are independent of alginate concentration and temperature. Lower absolute lp values were found for DMEM solutions compared with the other solvents. Moreover, scaling exponents for ηsp, G0, and TR do not change with temperature, within 20 and 60 °C. These findings suggest no change in the conformation of alginate chains with temperature.Graphical abstractAlginate rheological properties depend on the solvent quality

Meso-Substituent-Directed Aggregation Behavior and Water Solubility: Direct Functionalization of Methine Chain in Thiazole Orange and Biological Applications in Aqueous Buffer.

A new strategy is presented to preclude aggregation and enhance water solubility of cyanine dyes. Namely, a heteroatom-containing substituent, for distorting molecular plane and increasing interaction with water molecules, is introduced to the methine chain of 2-thiazole orange (1, a monocyanine) via one-step, and 2-thiazole orange derivatives 2a-g are prepared accordingly. The X-ray crystal structures show that the molecular plane of 2a-g is drastically twisted, which reduces intermolecular π-π stacking. The derivatives 2a-g exhibit good to excellent water solubility and can be dissolved in aqueous phosphate-buffered saline (PBS) at concentrations suitable for biomedical applications. No aggregation in aqueous PBS, relatively high molar extinction coefficients, and low solvatochromism of 2a-g are reflected by the UV-vis spectra. Compound 2b shows fast response and high selectivity for biothiols (Cys, Hcy, and GSH) in aqueous PBS and is further employed to detect endogenous biothiols with decent biocompatibility as demonstrated by live cell fluorescence imaging.

Efficient syntheses, crystal structure, thermal and biological evaluation of amlodipine 4-chlorobenzoyl, 4-chlorobenzene and 2,5-dichlorobenzene sulfonamide derivatives

An efficient synthesis of new A-2 , A-3 , and A-4 analogues from amlodipine ( A-1 ) has been achieved. All synthesized compounds were investigated by elemental analysis, FTIR, EIMS, and 1 H NMR techniques. Crystal structures of A-2 and A-3 were determined by single crystal X-ray diffraction method. Compound A-2 crystallizes in a monoclinic space group C 2/ c having unit cell parameters a = 23.8754(9) Å, b = 8.6725(3) Å, c = 30.5777(12) Å, β = 90.673(2)°, and V = 6331.0(4) Å 3 , whereas A-3 crystallizes in a triclinic space group P 1 ¯ having unit cell parameters a = 8.2968(3) Å, b = 9.3112(4) Å, c = 18.1359(7) Å, α = 100.692(2)°, β = 98.316(3)°, γ = 102.747(2)°, and V = 1317.39(9) Å 3 . These compounds showed that C–H⋯O and N–H⋯O hydrogen bonds stabilize the crystal packing. The results of thermal analysis of all products were consistent with the proposed stoichiometry and compounds were found thermally stable up to 200 °C. The compounds were tested for direct free radical scavenging effect toward α, α-Diphenyl-1-picryl hydrazide (DPPH • ) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS •+ ) radical cation in aqueous phosphate-buffered saline of pH 7.4 and showed significant in vitro antioxidant potential. Antiurease activity was also performed; A-2 and A-4 showed excellent results with dose independency.

Equilibrium water and solute uptake in silicone hydrogels

Equilibrium water content of and solute partitioning in silicone hydrogels (SiHys) are investigated using gravimetric analysis, fluorescence confocal laser-scanning microscopy (FCLSM), and back extraction with UV/Vis-absorption spectrophotometry. Synthesized silicone hydrogels consist of silicone monomer, hydrophilic monomer, cross-linking agent, and triblock-copolymer macromer used as an amphiphilic compatibilizer to prevent macrophase separation. In all cases, immiscibility of the silicone and hydrophilic polymers results in microphase-separated morphologies. To investigate solute uptake in each of the SiHy microphases, equilibrium partition coefficients are obtained for two hydrophilic solutes (i.e., theophylline and caffeine dissolved in aqueous phosphate-buffered saline) and two oleophilic solutes (i.e., Nile Red and Bodipy Green dissolved in silicone oil), respectively. Measured water contents and aqueous-solute partition coefficients increase linearly with increasing solvent-free hydrophilic-polymer volume fraction. Conversely, oleophilic-solute partition coefficients decrease linearly with rising solvent-free hydrophilic-polymer volume fraction (i.e., decreasing hydrophobic silicone-polymer fraction). We quantitatively predict equilibrium SiHy water and solute uptake assuming that water and aqueous solutes reside only in hydrophilic microdomains, whereas oleophilic solutes partition predominately into silicone microdomains. Predicted water contents and solute partition coefficients are in excellent agreement with experiment. Our new procedure permits a priori estimation of SiHy water contents and solute partition coefficients based solely on properties of silicone and hydrophilic homopolymer hydrogels, eliminating the need for further mixed-polymer-hydrogel experiments.

Pyrene-wired antibodies on highly oriented pyrolytic graphite as a label-free impedance biosensor for the sepsis biomarker procalcitonin

An antibody-based, label-free impedance biosensor for the sepsis biomarker procalcitonin (PCT) has been produced using highly oriented pyrolytic graphite (HOPG) as the sensor electrode material and with attachment of the PCT capture antibody via coupling to 1-pyrenebutanoic acid succinimidyl ester (PYSE) pre-immobilised on the electrode by π-π stacking. In an aqueous phosphate-buffered saline (PBS) at pH = 7.4, the charge-transfer resistance for the redox probe Ru(NH3)6 3+/2+ exhibited a linear dependence on the PCT concentration in the range 10 to 100 ng mL−1 and provided a detection limit for PCT of 3.70 ng mL−1. Also, over the same PCT concentration range, the electrode capacitance showed a steady decrease, offering the prospect of a capacitive sensing mode for the label-free device, possibly removing the need to add a redox species to the sample under test. These results demonstrate the feasibility of using pyrene-modified HOPG as a simple platform for label-free impedance biosensors, here for the important sepsis biomarker PCT.

Antioxidant Properties of Cultivars and Selections from the Colorado Potato Breeding Program

Consumers have become increasingly aware of potential health benefits from diets rich in fruits and vegetables. While potato has not yet surfaced as a headline-grabber in this respect, there is increasing evidence that some genotypes may possess health attributes that warrant attention. Plant breeders rely on germplasm biodiversity to advance their programs and are also acutely aware of current marketing trends that relate to health attributes. Investigations of antioxidant properties for over 90 genotypes were conducted to characterize antioxidant profiles for the Colorado potato breeding program and to identify those especially rich in antioxidants. The objective was to summarize data based on total phenolics (TP), Trolox equivalent antioxidant capacity (TEAC) and vitamin C, as well as to provide LC/MS characterization of major phenolic compounds and glycoalkaloids for pigmented genotypes. Preliminary data from breast cancer cell culture inhibition studies were examined for relationships to in vitro chemical assays. Genotypes with red or purple skin and flesh consistently had the highest gallic acid equivalent TP, TEAC, and chlorogenic acid content. Baked tubers had lower TP levels, TEAC and vitamin C compared to uncooked, microwave cooked and boiled tubers. Environmental effects contributed year to year variation in TP and TEAC radical scavenging capacity. TP content increased after 6 to 7 months in refrigerated storage at 5 ± 1°C in several highly pigmented, but not in white or yellow fleshed cultivars and selections. Major phenolic compounds, anthocyanins, and glycoalkaloid content were further investigated with LC/MS in six cultivars. The pigmented cultivars ‘Purple Majesty’ and ‘Mountain Rose’ contained considerably higher levels of chlorogenic acid isomers than the non-pigmented genotypes. In the non-pigmented genotypes, chlorogenic acid and glycolalkaloid content were highest in ‘Rio Grande Russet’. Chlorogenic acid has been demonstrated to exhibit several desirable anticarcinogenic properties in recent biochemical investigations, as have several of the phenolic based anthocyanin pigments found in many colorful fruits and vegetables. Thus, comprehensive compositional profiles using LC/MS are of interest in characterizing germplasm for breeding purposes. Preliminary tests with phosphate buffered saline (PBS) extracts of baked tubers from six cultivars revealed that ‘Rio Grande Russet’ was most effective in inhibiting growth of breast cancer cultures MCF-7 and MDA-MB-468. ‘Purple Majesty’ inhibited to some extent, ‘Mountain Rose’ and ‘Yukon Gold’ had no inhibitory effect. A subsequent study with 21 genotypes where the initial extract was made with 80% acetone followed by drying and extraction in aqueous PBS differed from direct PBS extraction. Five genotypes including ‘Russet Nugget’ inhibited at 0.187% to 0.375% w/v of the cell culture solution. However, IC50 inhibition data was not strongly related to the in vitro chemical data for these cultivars.

Interfacial Energetics of Protein Adsorption from Aqueous Buffer to Surfaces with Varying Hydrophilicity

Adsorption isotherms constructed from time-and-concentration-dependent advancing contact angles thetaa show that the profound biochemical diversity among ten different blood proteins with molecular weight spanning 10-1000 kDa has little discernible effect on the amount adsorbed from aqueous phosphate-buffered saline (PBS) solution after 1 h contact with a particular test surface selected from the full range of observable water wettability (as quantified by PBS adhesion tension tauoa=gammaolv cos thetaoa; where gammaolv is the liquid-vapor interfacial tension and thetaoa is the advancing PBS contact angle). The maximum advancing spreading pressure, Pimaxa, determined from adsorption isotherms decreases systematically with tauoa for methyl-terminated self-assembled monolayers (CH3 SAM, tauo=-15 mN/m), polystyrene spun-coated onto electronic-grade SiOx wafers (PS, tauo=7.2 mN/m), aminopropyltriethoxysilane-treated SiOx surfaces (APTES, tauo = 42 mN/m), and fully water wettable SiOx (tauo=72 mN/m). Likewise, the apparent Gibbs' surface excess [Gammasl-Gammasv], which measures the difference in the amount of protein adsorbed Gamma (mol/cm2) at solid-vapor (SV) and solid-liquid (SL) interfaces, decreases with tauo from maximal values measured on the CH3 SAM surface through zero (no protein adsorption in excess of bulk solution concentration) near tauo=30 mN/m (thetaa=65 degrees). These latter results corroborate the conclusion drawn from independent studies that water is too strongly bound to surfaces with tauo>or=30 mN/m to be displaced by adsorbing protein and that, as a consequence, protein does not accumulate within the interfacial region of such surfaces at concentrations exceeding that of bulk solution ([Gammasl-Gammasv]=0 at tauo=30 mN/m). Results are collectively interpreted to mean that water controls protein adsorption to surfaces and that the mechanism of protein adsorption can be understood from this perspective for a diverse set of proteins with very different amino acid compositions.

Determination of toxic carbonyl compounds in cigarette smoke

Toxic carbonyl compounds, including formaldehyde, malonaldehyde, and glyoxal, formed in mainstream cigarette smoke were quantified by derivatization-solid phase extraction-gas chromatography methods. Cigarette smoke from 14 commercial brands and one reference (2R1F) was drawn into a separatory funnel containing aqueous phosphate-buffered saline. Reactive carbonyl compounds trapped in the buffer solution were derivatized into stable nitrogen containing compounds (pyrazoles for beta-dicarbonyl and alpha,beta-unsaturated aldehyde; quinoxalines for alpha-dicarbonyls; and thiazolidines for alkanals). After derivatives were recovered using C(18) solid phase extraction cartridges, they were analyzed quantitatively by a gas chromatograph with a nitrogen phosphorus detector. The total carbonyl compounds recovered from regular size cigarettes ranged from 1.92 mg/cigarette(-1) to 3.14 mg/cigarette(-1). The total carbonyl compounds recovered from a reference cigarette and a king size cigarette were 3.23 mg/cigarette(-1) and 3.39 mg/cigarette(-1), respectively. The general decreasing order of the carbonyl compounds yielded was acetaldehyde (1110-2101 microg/cigarette(-1)) > diacetyl (301-433 microg/cigarette(-1)), acrolein (238-468 microg/cigarette(-1)) > formaldehyde (87.0-243 microg/cigarette(-1)), propanal (87.0-176 microg/cigarette(-1)) > malonaldehyde (18.9-36.0 microg/cigarette(-1)), methylglyoxal (13.4-59.6 microg/cigarette(-1)) > glyoxal (1.93-6.98 microg/cigarette(-1)).

Conformational mapping of the N-terminal peptide of HIV-1 gp41 in lipid detergent and aqueous environments using 13C-enhanced Fourier transform infrared spectroscopy.

The N-terminal domain of HIV-1 glycoprotein 41,000 (gp41) participates in viral fusion processes. Here, we use physical and computational methodologies to examine the secondary structure of a peptide based on the N terminus (FP; residues 1-23) in aqueous and detergent environments. (12)C-Fourier transform infrared (FTIR) spectroscopy indicated greater alpha-helix for FP in lipid-detergent sodium dodecyl sulfate (SDS) and aqueous phosphate-buffered saline (PBS) than in only PBS. (12)C-FTIR spectra also showed disordered FP conformations in these two environments, along with substantial beta-structure for FP alone in PBS. In experiments that map conformations to specific residues, isotope-enhanced FTIR spectroscopy was performed using FP peptides labeled with (13)C-carbonyl. (13)C-FTIR results on FP in SDS at low peptide loading indicated alpha-helix (residues 5 to 16) and disordered conformations (residues 1-4). Because earlier (13)C-FTIR analysis of FP in lipid bilayers demonstrated alpha-helix for residues 1-16 at low peptide loading, the FP structure in SDS micelles only approximates that found for FP with membranes. Molecular dynamics simulations of FP in an explicit SDS micelle indicate that the fraying of the first three to four residues may be due to the FP helix moving to one end of the micelle. In PBS alone, however, electron microscopy of FP showed large fibrils, while (13)C-FTIR spectra demonstrated antiparallel beta-sheet for FP (residues 1-12), analogous to that reported for amyloid peptides. Because FP and amyloid peptides each exhibit plaque formation, alpha-helix to beta-sheet interconversion, and membrane fusion activity, amyloid and N-terminal gp41 peptides may belong to the same superfamily of proteins.

Heat Resistance of Acanthamoeba sp. Cysts in Green Mussel Broth and Phosphate-Buffered Saline

Cysts propagated by the agar block method with heat-killed Escherichia coli as nutrient overlay were inoculated into Perna viridis broth (PVB) and phosphate-buffered saline (PBS) prior to exposure to 60, 75 and 100°C for 0, 3, 5, and 10 min. The heat resistance of Acanthamoeba sp. cysts expressed in of D- and Z- values were found to be greater in the complex organic PVB than in the aqueous PBS. The established D- values in the PVB were 81.20, 44.59, and 8.83 min at 60, 75, and 100°C, respectively. The calculated Z-value of Acanthamoeba sp. cysts in PVB was 40.28°C.

Buccal permeation of [ d-Ala 2, d-Leu 5]enkephalin from liquid crystalline phases of glyceryl monooleate

The ex vivo buccal permeability of a [ d-Ala 2, d-Leu 5]enkephalin (DADLE) and glyceryl monooleate (GMO) was examined from the cubic and lamellar liquid crystalline phases of GMO and aqueous phosphate-buffered saline (pH 7.4, PBS) solution across excised porcine buccal mucosa mounted in a Franz cell. GMO was released in vitro from the liquid crystalline phases indicating the erosion of the liquid crystal matrices. GMO released from the liquid crystalline matrices permeated the porcine buccal mucosa with fluxes of 0.10±0.03 and 0.07±0.00%/cm 2 per h for the cubic and lamellar phases, respectively. The flux of DADLE (1.21±0.32 and 1.15±0.11%/cm 2 per h for the cubic and lamellar phases, respectively) from the liquid crystalline phases was significantly enhanced by the GMO compared with PBS solution (0.43±0.08%/cm 2 per h) during the initial permeation phase ( t<3 h). Our results suggest that the cubic and lamellar liquid crystalline phases can be considered as promising buccal drug carriers for peptide drugs as well as acting as permeation enhancers.

Effects of protein-surface interactions on protein ion signals in MALDI mass spectrometry.

The influence of polymer surface-protein binding affinity on protein ion signals in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is examined. The surfaces of poly(vinylidene fluoride) and poly(ethylene terephthalate) polymer substrates are modified by pulsed rf plasma deposition of allylamine. By varying the on/off duty cycle of the pulsed rf plasma, the polymer substrate surfaces are coated with thin films having varying densities of surface amine groups. The varying surface amine density is shown to lead to systematic changes in the surface binding affinity for the 125I-radiolabeled peptides angiotensin I and porcine insulin. Unlabeled angiotensin I and porcine insulin are then deposited on the pulsed rf plasma-modified substrates and analyzed by MALDI mass spectrometry. The experimental approach involves applying the peptide to the modified polymer surface in an aqueous phosphate-buffered saline solution and allowing the peptide solution to dry completely under ambient conditions. Subsequently, the MALDI matrix alpha-cyano-4-hydroxycinnamic acid in methanol and 10% trifluoroacetic acid in water are added to the peptide-coated modified polymer surfaces. The results of these studies demonstrate that, for the sample preparation method employed, increases in the surface peptide binding affinity lead to decreases in the peptide MALDI ion signal.

Delayed fluorescence of porphyrins in different media

Lifetimes of TPPS4 (meso-tetraphenylporphine tetrasulfonate) triplet states were measured for liquid solutions of different acidity and viscosity and as a function of acceptor concentration for different acceptors (bovine serum albumin, tryptophan and furfuryl alcohol). Triplet lifetimes were estimated by monitoring the decay of TPPS4 delayed fluorescence of E-type. The lifetime of delayed fluorencence depends on the concentration of O2, since the latter is an effective quencher of the triplet state. The lifetime is shown to be influenced mainly by degree of aggregation state of TPPS4 and, therefore, by the pH of the solution, decreasing with pH and for each pH remaining constant over a wide range of acceptor concentrations. The monomeric species is found to have the longest triplet lifetime in aqueous phosphate-buffered saline solution at neutral pH, especially when bound to albumin, despite of the low viscosity and protonated nature of the medium.

The effect of solvent deuteration on the photophysics of sulphonated aluminium phthalocyanine

The photophysics of disulphonated aluminium phthalocyanine in protiated and deuterated aqueous phosphate-buffered saline solution are reported. In the deuterated solvent the rate coefficients of fluorescence and intersystem crossing are unaffected by the heavier isotope; however, the rate of internal conversion from S 1 to S 0 has been shown to be considerably reduced. This results in increased quantum yields of fluorescence and triplet-state formation and also increased triplet and singlet lifetimes in deuterium oxide (Φ f0.40±0.04, τ f5.1±0.1 ns and τ t600±30 μs in H 2O and Φ f0.52±0.06, τ f6.8±0.1 ns and τ t1590±100 μs in D 2O). The implications of this with respect to the determination of the quantum yields of singlet oxygen formation in D 2O are discussed.

Immunochemical Studies of Infectious Mononucleosis

Abstract A glycoprotein was isolated from goat erythrocyte membranes by extraction with hot 75% ethanol. The glycoprotein was purified by ethanol precipitation, phosphocellulose chromatography gel filtration, ethyl:ether and chloroform:methanol extraction. In aqueous phosphate-buffered saline, pH 7, the glycoprotein was in an aggregated state with a sedimentation coefficient (Sobs) of 21. Addition of 0.5% sodium dodecyl sulphate (SDS) lowered Sobs to 1.5. Electrophoresis of the glycoprotein on polyacrylamide gels containing phosphate-buffered 0.1% SDS gave a single band, staining with both periodic acid Schiff (PAS) and Coomassie Blue (CB). The apparent m.w., calculated from retardation coefficient, was 25,000. Electrophoresis of the glycoprotein on 1% SDS gels buffered with Tris-acetate (pH 7.4) showed a major band of similar (23,500) apparent m.w. plus four other PAS- and CB-staining bands of lower mobility. With 131I-labeled glycoprotein, recovery of bands from gels, sialic acid analysis, heterophile antigen activity, and re-electrophoresis, it was shown that these additional bands were aggregated forms of a single or closely related glycoprotein species. The purified glycoprotein contained 50% carbohydrate with molar ratios of sialic acid: galactose: mannose: galactosamine: glucosamine of 3.1:2.1:0.1:1.6:1. The glycoprotein was highly reactive with the Paul-Bunnell heterophile antibody in the sera of patients with infectious mononucleosis, with Limulus polyphemus lectin and weakly ractive with wheat germ agglutinin. These reactivities were destroyed by neuraminidase treatment or by alkaline sodium borohydride. The native glycoprotein did not react with lectins from Canavalia enisformis, Phaseolus vulgaris, Ricinus communis, or Vicia graminea although it was reactive with the latter two after neuraminidase treatment.