Design and development of a fiber-optic immunosensor utilizing near-infrared fluorophores

The design and application of a fluorescent fiber-optic immunosensor (FFOI) are reported. The FFOI is utilized for the detection of antibody/antigen binding within the near-infrared (NIR) spectral region. The technique is developed through the combined use of fiber-optic, semiconductor laser-excitation, fluorescence detection, NIR dye, and immunochemical techniques. The antibody is immobilized on the FFOI and utilized as a recognition component for trace amounts of specific antigen. The FFOI is constructed to utilize an antibody sandwich technique. The assay involves the immobilization of the capture antibody on the sensing tip of the FFOI followed by the exposure of the immobilized sensing tip to the antigen. The antigen-coated FFOI is then introduced to a second antibody previously labeled with the NIR dye. Typical measurements are performed in about 15 min. A semiconductor laser provides the excitation (780 nm) of the immune complex. The resulting emission is detected by a silicon photodiode detector (820 nm). The intensity of the resulting fluorescence is directly proportional to the concentration of the antigen. The sensitivity of the analysis reaches 10 ng/ml and the response time is 10–15 min.     

NMR and EPR structural delineation of copper(II) complexes formed by kanamycin A in water

The complexes formed by kanamycin A at three different pH values (5.5, 7.4 and 12.0) were investigated by NMR and EPR spectroscopy. Paramagnetic relaxation contributions to proton relaxation rates were measured using a combination of the TOCSY sequence with the inversion recovery experiment in order to gain signal resolution in the bulk region. Measured contributions were converted into distances and used for structural determination by restrained simulated annealing where all possible chair and boat conformations of the rings were taken into account. The interaction of the Cu(II) ion with the nitrogen of the C ring is apparent at all pH values. At higher pH also the amino group of ring A starts to be involved in the metal coordination sphere. This is accompanied by a switch in conformation of ring C. Structures are consistent with the involvement in the coordination sphere either of the 2' or 4' hydroxyl oxygens at pH 5.5 and the 5 and the 6' hydroxyl oxygens (or the ring oxygen) at pH 12.0.     

Combining cationic ring‐opening polymerization and click chemistry for the design of functionalized polyurethanes

A straightforward strategy for the synthesis and functionalization of polyurethanes (PUs) via the use of alkyne‐functionalized polytetrahydrofuran (PTHF) diols is described. The alkyne groups have been introduced into the PTHF chains by the cationic ring‐opening copolymerization of tetrahydrofuran and glycidyl propargyl ether. These PTHF prepolymers were combined with 1,4‐butanediol and hexamethylene diisocyanate for the synthesis of linear PUs with latent functionalization sites. The polyether segments of the PUs have then been coupled with several types of functionalized azides by the copper‐catalyzed azide‐alkyne “click” chemistry, for example with phosphonium containing azides for their antibacterial properties. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Influence of (phospho)lipases on properties of mica supported phospholipid layers

The effect of enzymes: lipase from Candida cylindracea (L_Cc), phospholipase A₂ from hog pancreas (PLA₂) and phospholipase C from Bacillus cereus (PLC) to modulate wetting properties of solid supported phospholipid bilayers was studied via advancing and receding contact angle measurements of water, formamide and diiodomethane, and calculation of the surface free energy and its components from van Oss et al. (LWAB) and contact angle hysteresis (CAH) approaches. Simultaneously, topography of the studied layers was determined by Atomic Force Microscopy (AFM). The investigated lipid bilayers were transferred on mica plates from subphase of pure water by means of Langmuir-Blodgett and Langmuir-Schaefer techniques. The investigated phospolipid layers were: saturated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), unsaturated DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and their mixture DPPC/DOPC. The obtained results revealed that the lipid membrane degradation by the enzymes caused increase in its surface free energy due to the amphiphilic hydrolysis products, which may accumulate in the lipid bilayer. In result activity of the enzymes may increase and then break down the bilayer structure takes place. It is likely that after dissolution of the hydrolysis reaction products in the bulk phase, patches of bare mica surface are accessible, which contribute to the apparent surface free energy changes. Comparison of AFM images and the free energy changes of the layers gives better insight into changes of their properties. The observed gradual increase in the layer surface free energy allows controlling of the hydrolysis process to obtain the surfaces of defined properties.     

A novel hydroxy functionalized polyester obtained by ring opening copolymerization of L‐lactide with a pyrolysis product of cellulose

A hydroxylactone ((1R,5S)‐1‐hydroxy‐3,6‐dioxabicyclo [3.2.1] octan‐2‐one, abbreviated as LAC) obtained from catalytic pyrolysis of cellulose was investigated as a monomer in the synthesis of polyesters by ring‐opening polymerization (ROP) with L ‐lactide. Although stannous octoate resulted inactive, ROP initiated by zirconium (IV) acetylacetonate afforded novel copolyesters from LAC and lactide mixtures in the bulk at 110 °C. Copolymers were obtained with different LAC content (from 19 to 45%) with a random microstructure as established by detailed NMR analysis. FTIR spectrometry confirmed the presence in the polymer chain of the OH groups originally present in LAC, which do not react during polymerization due to steric hindrance and inter/intramolecular hydrogen bonding. Reaction with trichloroacetylisocyanide proved that OH groups of the polyesters can be readily derivatized. The application of LAC as a comonomer enables the insertion of the alcohol functionality in polylactide avoiding protection/deprotection steps and potentially expanding the realm of biomaterials affordable from carbohydrate feedstock. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 247–257, 2009     

Are Gait and Balance Problems in Neurological Patients Interdependent? Enhanced Analysis Using Gait Indices,Cyclograms, Balance Parameters and Entropy

Background: Balance and locomotion are two main complex functions, which require intact and efficient neuromuscular and sensory systems, and their proper integration. In many studies the assumption of their dependence is present, and some rehabilitation approaches are based on it. Other papers undermine this assumption. Therefore the aim of this study was to examine the possible dependence between gait and balance in patients with neurological or sensory integration problems, which affected their balance. Methods: 75 patients (52 with neurological diseases, 23 with sensory integration problems) participated in the study. They underwent balance assessment on Kistler force plate in two conditions, six tests on a Balance Biodex System and instrumented gait analysis with VICON. The gait and balances parameters and indices, together with entropy and cyclograms were used for the analysis. Spearman correlation, multiple regression, cluster analysis, and discriminant analysis were used as analytical tools. Results: The analysis divided patients into 2 groups with 100% correctly classified cases. Some balance and gait measures are better in the first group, but some others in the second. Conclusions: This finding confirms the hypothesis that there is no direct link between gait and balance deficits.     

In situ detection of a single carotenoid crystal in a plant cell using Raman microspectroscopy

It is the first report of direct, in situ detection of carotenoids at the subcellular level by using Raman microspectroscopy. Single crystals sequestered in a carrot cell were measured using FT-Raman spectrometer equipped with a microscope and 40× objective. The observed characteristic bands centered at 1518 cm⁻¹ and 1156 cm⁻¹ proved the crystals were composed of carotenoids with β-carotene being predominant. The obtained results show the potential of Raman microspectroscopy for identification and analysis of compounds localized in cytoplasm by taking measurements directly from a single plant cell.     

Inclusion Complex Formation Between Modified Cyclodextrins and Riboflvin and Alloxazine in Aqueous Solution

Inclusion complex formation of hydroxypropylated α-, β- and γ-cyclodextrins with riboflavin (vitamin B₂) and alloxazine was studied by spectroscopic and solubility methods. Alloxazine, which is a structural analog of riboflavin, was considered in order to evaluate the role of ribityl and methyl substituents in complexation. Thermodynamic parameters for 1:1 complex formation were obtained and analyzed in terms of influence of the reagent structure on the binding process. It was shown that the cavity of hydroxypropyl-β-cyclodextrin is more appropriate for formation of stable complexes. The complexes are enthalpy stabilized, due to prevalence of van der Waals interactions and possible hydrogen bonding. The partial insertion of riboflavin into the cyclodextrin cavity was revealed by ¹H NMR and computer modeling. The ribityl side chain, which prevents deep inclusion, is located nearby the wider rim of the cyclodextrin molecule and can undergo destruction. Penetration of the alloxazine molecule into the macrocyclic cavity is deeper and accompanied by formation of more stable inclusion complexes. Hydroxypropyl-β-cyclodextrin was found to be the more efficient solubilizing agent for riboflavin and alloxazine, whereas a stabilization action of cyclodextrins towards riboflavin was not observed.     

A novel approach to the rapid determination of amoxicillin in human plasma by solid phase microextraction and liquid chromatography

A new approach to the rapid determination of amoxicillin (AMO) in human plasma followed by solid phase microextraction (SPME) fiber coatings based on conducting polymers (polypyrrole and polythiophene) and high performance liquid chromatography (HPLC) has been described. The porous structures of the electrochemically deposited polymer coatings have been characterized by scanning electron microscopy (SEM). The experimental parameters relating to the extraction efficiency of the SPME fibers such as pH, extraction time and desorption conditions (solvents, time) were studied and selected. The SPME/HPLC-UV method was linear over a working range of 1-50 μg ml(-1). The inter-day accuracy (expressed as coefficients of variations, CVs) was less than 15% and precision (expressed as the relative standard deviations, RSDs) with percentage values was less than 5.9%. Amoxicillin was found to be stable in the human plasma at room temperature (20 °C) within 8 hours. The developed method was successfully applied to the analysis of real human plasma samples. The limit of detection and limit of quantification for amoxicillin in plasma were 1.21 μg ml(-1) and 3.48 μg ml(-1), respectively.