Autofluorescence characterization of advanced glycation end products of hemoglobin

This article describes the analysis of autofluorescence of advanced glycation end products of hemoglobin (Hb-AGE). Formed as a result of slow, spontaneous and non-enzymatic glycation reactions, Hb-AGE possesses a characteristic autofluorescence at 308/345 nm (lambda(ex)/lambda(em)). Even in the presence of heme as a quenching molecule, the surface presence of the glycated adduct gave rise to autofluorescence with the quantum yield of 0.19. The specificity of monoclonal antibody developed against common AGE structure with Hb-AGE was demonstrated using reduction in fluorescence polarization value due to increased molecular volume while binding. The formation of fluorescent adduct in hemoglobin in the advanced stage of glycation and the non-fluorescent HbA(1c) will be of major use in distinguishing and to know the past status of diabetes mellitus. While autofluorescence correlated highly with HbA(1c) value under in vivo condition (r = 0.85), it was moderate in the clinical samples (r = 0.55). The results suggest a non-linear relation between glycemia and glycation, indicating the application of Hb-AGE as a measure of susceptibility to glycation rather than glycation itself.     

Fluorescence and biochemical characterization of glycated hemoglobin

Isoelectric focusing (IEF) of glycated hemoglobin (GHb) was carried out in ultra-thin polyacrylamide gels to separate the hemoglobin-advanced glycation endproducts (Hb-AGEs) from the hemoglobin-A_1C (HbA_1C) fraction. Precast polyacrylamide gels (Ampholine^® PAGplate) were used in Pharmacia LKB Multiphor II for this purpose. The separated bands for Hb-AGE and HbA_1C based on their isoelectric point (pI), were confirmed with the purifed fractions obtained from the cation exchange chromatographic technique. From the calibration curve, the pI values were found to be 6.748 and 6.495 for HbA_1C and Hb-AGE, respectively. The lowering of pI values for glycated hemoglobin, when compared to unglycated hemoglobin (pI = 6.852), can be attributed to the glycation at the amino terminals of the peptide chains. Increased reduction in pI value for Hb-AGE can be attributed to the effect of glycation of amino groups at various sites on the peptide chains, apart from the terminal amino groups. Fluorescence analysis was carried out for the purified fraction of Hb-AGE which showed the formation of a new fluorophor adduct having the excitation and emission maxima at 308 nm and 345 nm, respectively. Time-dependent formation of Hb-AGE under in vitro conditions was monitored by fluorescence (308/345 nm) over a period of 120 days, which showed its formation only after 3 weeks of incubation.     

A microfluidic electrochemical device for high sensitivity biosensing: Detection of nanomolar hydrogen peroxide

We report herein a simple device for rapid biosensing consisting of a single microfluidic channel made from poly(dimethylsiloxane) (PDMS) coupled to an injector, and incorporating a biocatalytic sensing electrode, reference and counter electrodes. The sensing electrode was a gold wire coated with 5 nm glutathione-decorated gold nanoparticles (AuNPs). Sensitive detection of H₂O₂ based on direct bioelectrocatalysis by horseradish peroxidase (HRP) was used for evaluation. HRP was covalently linked the glutathione–AuNPs. This electrode presented quasi-reversible cyclic voltammetry peaks at ?0.01 V vs. Ag/AgCl at pH 6.5 for the HRP heme Fe^III/Fe^II couple. Direct electrochemical activity of HRP was used to detect H₂O₂ at high sensitivity with a detection limit of 5 nM in an unmediated system.     

A membrane-less enzymatic fuel cell with layer-by-layer assembly of redox polymer and enzyme over graphite electrodes

Layer-by-layer (LBL) assembly of alternate osmium redox polymers and glucose oxidase, at anode, and laccase, at cathode, using graphite electrodes form a membrane-less glucose/O(2) enzymatic fuel cell providing a power density of 103 μW cm(-2) at pH 5.5.     

Spectroscopic characterization of zinc oxide nanorods synthesized by solid-state reaction

Well-crystallized zinc oxide nanorods have been fabricated by single step solid-state reaction using zinc acetate and sodium hydroxide, at room temperature. The sodium lauryl sulfate (SLS) stabilized zinc oxide nanorods were characterized by using X-ray diffraction, Fourier transform infrared spectroscopy, transmission electron microscopy and photoluminescence spectroscopy. The X-ray diffraction revealed the wurtzite structure of zinc oxide. The size estimation by XRD and TEM confirmed that the ZnO nanorods are made of single crystals. The growth of zinc oxide crystals into rod shape was found to be closely related to its hexagonal nature. The mass ratio of SLS:ZnO in the nanorods was found to be 1:10 based on the thermogravimetric analysis. Blue shift of photoluminescence emission was noticed in the ZnO nanorods when compared to that of ZnO bulk. FT-IR analysis confirmed the binding of SLS with ZnO nanorods. Apart from ease of preparation, this method has the advantage of eco-friendliness since the solvent and other harmful chemicals were eliminated in the synthesis protocol.     

Extraction of discontinuous structure-activity relationships from compound data sets through particle swarm optimization

The characterization of structure-activity relationship (SAR) features of large compound data sets has been a hot topic in recent years, and different methods for large-scale SAR analysis have been introduced. The exploration of local SAR components and prioritization of compound subsets have thus far mostly relied on graphical analysis methods that capture similarity and potency relationships in a systematic manner. A currently unsolved problem in large-scale SAR analysis is how to automatically select those compound subsets from large data sets that carry most SAR information. For this purpose, we introduce a numerical optimization scheme that is based on particle swarm optimization guided by an SAR scoring function. The methodology is applied to four large compound sets. We demonstrate that compound subsets representing the most discontinuous local SARs are consistently selected through particle swarm optimization.     

Silver-protein (core-shell) nanoparticle production using spent mushroom substrate

A simple route for the synthesis of silver-protein (core-shell) nanoparticles using spent mushroom substrate (SMS) has been demonstrated in this work. SMS exhibits an organic surface that reduces silver ions and stabilizes the silver nanoparticles by a secreted protein. The silver nitrate solution incubated with SMS changed to a yellow color from 24 h onward, indicating the formation of silver nanoparticles. The purified solution yielded the maximum absorbance at 436 nm due to surface plasmon resonance of the silver nanoparticles. X-ray analysis of the freeze-dried powder of silver nanoparticles confirmed the formation of metallic silver. Transmission electron microscopic analysis of the samples showed a uniform distribution of nanoparticles, having an average size of 30.5 +/- 4.0 nm, and its corresponding electron diffraction pattern confirmed the face-centered cubic (fcc) crystalline structure of metallic silver. The characteristic fluorescence of the protein shell at 435 nm was observed for the silver nanoparticles in solution, when excited at 280 nm, while Fourier transform infrared (FTIR) spectroscopy confirmed the presence of a protein shell. The silver nanoparticles were found to be stable in solution for more than 6 months. It is observed that the reducing agents from the safflower stalks caused the reduction of silver ions while protein secreted by the fungus stabilized the silver nanoparticles. These silver nanoparticles showed excellent antibacterial activity against two representative bacteria, Staphylococcus aureus (Gram positive) and Klebsiella pneumoniae (Gram negative), in spite of the presence of an organic layer as a shell. Apart from ecofriendliness and easy availability, "SMS" as a biomanufacturing unit will give us an added advantage in ease of handling when compared to other classes of microorganisms.     

The effect of fiber orientation and stacking sequence on carbon/E-glass/epoxy intraply hybrid composites under dynamic loading conditions

This study investigated the dynamic mechanical properties of hybrid intraply carbon/E-glass epoxy composites with different orientations and stacking sequences under different loading conditions with increasing temperature. A neat epoxy and five various hybrid composites such as Carbon (0°)/E-glass (90°), Carbon (45°)/E-glass (135°), Carbon (90°)/E-glass (0°), Carbon/E-glass (alternating layer), and Carbon/E-glass (alternating layer 45°) were manufactured. Three-point bending test and dynamic mechanical test were conducted to understand the flexural modulus and viscoelastic behavior (storage modulus, loss modulus, and loss tangent) of the composites. Dynamic mechanical test was performed with the dual cantilever method, at four different frequencies (1, 5, 10, and 20 Hz) and temperatures ranging from 30 to 150°C. The experimental results of storage modulus, loss modulus, and loss tangents were compared with the theoretical findings of neat epoxy and various hybrid composites. The glass transition temperature (T_g) increased with the increase in frequency. A linear fit of the natural log of frequency to the inverse of absolute temperature was plotted in the activation energy estimation. The interphase damping (tanδ_i) between plies and the strength indicator (S_i) of the hybrid composites were estimated. It was observed that the neat epoxy had more insufficient storage and loss modulus and a high loss tangent at all the frequencies whereas hybrid composites had high storage and loss modulus and a low loss tangent for all the frequencies. Compared with other hybrid composites, Carbon (90°)/E-glass (0°) had higher strength and activation energy. The result of reinforcement of hybrid fiber in neat epoxy significantly increases the material's strength and stability at higher temperatures whereas decreasing free molecular movement.