Determination of glycated hemoglobin with special emphasis on biosensing methods

The glycated hemoglobin (HbA1c) level in blood is a measure of long-term glycemic status in patients with diabetes mellitus. Current clinical methods for determination of the HbA1c level include electrophoresis/electroendosmosis, ion exchange chromatography, high-performance liquid chromatography, boronate affinity chromatography, immunoassay, and liquid chromatography–tandem mass spectroscopy in addition to fluorometry and colorimetry. These methods have certain drawbacks such as being complex, time-consuming, and requiring expensive apparatus and trained persons to operate. These drawbacks were overcome by biosensing methods. We review these biosensors, which are based on (i) measurement of electrons, that is, current generated from splitting of hydrogen peroxide released during oxidation of fructosyl valine by immobilized fructosyl amino acid oxidase, which is directly proportional to HbA1c concentration, and (ii) direct measurement of HbA1c by some specific reaction. HbA1c biosensors work optimally within 4 to 1800 s, between pH 7.0 and 9.0 and between 25 and 45 °C, and in the range of 1 to 10,000 μM, with a detection limit between 20 and 500 μM and sensitivity between 4.6 nA and 21.5 μA mM⁻¹ cm⁻² and stable over a period of 5 to 90 days. We suggest the ways to modify existing HbA1c biosensors, leading to simple, reliable, and economical sensors ideally suited for point-of-care treatment.     

Synthesis and characterization of a molecularly imprinted polymer and its application as SPE enrichment sorbent for determination of trace methimazole in pig samples using HPLC-UV

A novel molecularly imprinted polymer that could be applied as enrichment sorbent was prepared using methimazole (MMZ) as the template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker. Though evaluated by static, kinetic and competitive adsorption tests, the polymer exhibited high adsorption capacity, fast kinetics and good selective ability. A method for determination of trace MMZ was developed using this polymer as enrichment sorbent coupled with high performance liquid chromatography focusing on complex biological matrices. Under the optimum experimental conditions, the MMZ standard is linear within the concentration range studied, that is, from 0.5 μg L⁻¹ to 150 μg L⁻¹ (r² = 0.9941). Lower limits of detection (LOD, at S/N = 3) and quantification (LOQ, at S/N = 10) in pig samples were 0.63 μg kg⁻¹ and 2.10 μg kg⁻¹ for kidney, 0.51 μg kg⁻¹ and 1.70 μg kg⁻¹ for liver, 0.56 μg kg⁻¹ and 1.86 μg kg⁻¹ for muscle, respectively. Recoveries and relative standard deviation (RSD, n = 9) values for precision in the developed method were from 71.14% to 88.41% and from 2.53% to 6.18%.     

Effects on capacitance by overexpression of membrane proteins

Functional Channelrhodopsin-2 (ChR2) overexpression of about 10⁴ channels/μm² in the plasma membrane of HEK293 cells was studied by patch-clamp and freeze-fracture electron microscopy. Simultaneous electrorotation measurements revealed that ChR2 expression was accompanied by a marked increase of the area-specific membrane capacitance (C_m). The C_m increase apparently resulted partly from an enlargement of the size and/or number of microvilli. This is suggested by a relatively large C_m of 1.15 ± 0.08 μF/cm² in ChR2-expressing cells measured under isotonic conditions. This value was much higher than that of the control HEK293 cells (0.79 ± 0.02 μF/cm²). However, even after complete loss of microvilli under strong hypoosmolar conditions (100 mOsm), the ChR2-expressing cells still exhibited a significantly larger C_m (0.85 ± 0.07 μF/cm²) as compared to non-expressing control cells (0.70 ± 0.03 μF/cm²). Therefore, a second mechanism of capacitance increase may involve changes in the membrane permittivity and/or thickness due to the embedded ChR2 proteins.     

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14 ± 0.04 bonds/μm at 0.2 dyn/cm² to 0.014 ± 0.003 bonds/μm at 1.0 dyn/cm². In contrast, L-selectin tether bond formation increased from 0.017 ± 0.005 bonds/μm at 0.2 dyn/cm² to 0.031 ± 0.005 bonds/μm at 1.0 dyn/cm². L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.     

Examination of microbial fuel cell start-up times with domestic wastewater and additional amendments

Rapid startup of microbial fuel cells (MFCs) and other bioreactors is desirable when treating wastewaters. The startup time with unamended wastewater (118 h) was similar to that obtained by adding acetate or fumarate (110-115 h), and less than that with glucose (181 h) or Fe(III) (353 h). Initial current production took longer when phosphate buffer was added, with startup times increasing with concentration from 149 h (25 mM) to 251 h (50 mM) and 526 h (100 mM). Microbial communities that developed in the reactors contained Betaproteobacteria, Acetoanaerobium noterae, and Chlorobium sp. Anode biomass densities ranged from 200 to 600 μg/cm² for all amendments except Fe(Ш) (1650 μg/cm²). Wastewater produced 91 mW/m², with the other MFCs producing 50 mW/m² (fumarate) to 103 mW/m² (Fe(III)) when amendments were removed. These experiments show that wastewater alone is sufficient to acclimate the reactor without the need for additional chemical amendments.     

Magnetic single-enzyme nanoparticles with high activity and stability

Magnetic single-enzyme nanoparticles (SENs) encapsulated within a composite inorganic/organic polymer network were fabricated via the surface modification and in situ aqueous polymerization of separate enzyme molecule. The resultant nanoparticles were characterized by transmission electron microscope (TEM), Fourier transform infrared (FTIR) spectrometer and X-ray diffraction (XRD). These particles are almost spherical in shape and have a unique size of about 50 nm in diameter. Electrical and magnetic measurements reveal that the magnetic SENs have a conductivity of 2.7 × 10⁻³ S cm⁻¹, and are superparamagnetic with a saturation magnetization of 14.5 emu g⁻¹ and a coercive force of 60 Oe. Compared with free enzyme, encapsulated enzyme exhibits a strong tolerance to the variation of solution pH, high temperature, organic solvent and long-term storage, thus showing significantly enhanced enzyme performance and stability.     

Comparative evaluation of vacuum-based surface sampling methods for collection of Bacillus spores

In this study, four commonly-used sampling devices (vacuum socks, 37 mm 0.8 μm mixed cellulose ester (MCE) filter cassettes, 37 mm 0.3 μm polytetrafluoroethylene (PTFE) filter cassettes, and 3M™ forensic filters) were comparatively evaluated for their ability to recover surface-associated spores. Aerosolized spores (~ 10⁵ CFU cm^− 2) of a Bacillus anthracis surrogate were allowed to settle onto three material types (concrete, carpet, and upholstery). Ten replicate samples were collected using each vacuum method, from each material type. Stainless steel surfaces, inoculated simultaneously with test materials, were sampled with pre-moistened wipes. Wipe recoveries were utilized to normalize vacuum-based recoveries across trials. Recovery (CFU cm^− 2) and relative recovery (vacuum recovery/wipe recovery) were determined for each method and material type. Recoveries and relative recoveries ranged from 3.8 × 10³ to 7.4 × 10⁴ CFU cm^− 2 and 0.035 to 1.242, respectively. ANOVA results indicated that the 37 mm MCE method exhibited higher relative recoveries than the other methods when used for sampling concrete or upholstery. While the vacuum sock resulted in the highest relative recoveries on carpet, no statistically significant difference was detected. The results of this study may be used to guide selection of sampling approaches following biological contamination incidents.     

Antifeedant activity of ethanolic extract from Flourensia oolepis and isolation of pinocembrin as its active principle compound

The ethanolic extract from Flourensia oolepis aerial parts showed strong antifeedant activity against the pest larvae, Epilachna paenulata, with an antifeedant index (AI%) of 99.1% at 100 μg/cm². Based on chromatographic fractionation of the extract, guided by bioassays on E. paenulata, the flavanone pinocembrin (1) was isolated as the most active principle. In a choice assay, compound 1 showed strong antifeedant activity against E. paenulata, Xanthogaleruca luteola and Spodoptera frugiperda with an AI% of 90, 94 and 91% (p < 0.01) respectively, at 50 μg/cm². The dosages necessary for 50% feeding inhibition of the insects (ED₅₀) were 7.98, 6.13 and 8.86 μg/cm², respectively. The feeding inhibitory activity of 1 against E. paenulata was compared with the activity of other structurally related flavonoids like naringenin, which was inactive up to 100 μg/cm², catechin which was nearly 6 times less active than 1, and quercetin which was equally active as 1. The effect of these on the feeding behavior of E. paenulata was also studied.     

Topical anti-inflammatory activity of boropinic acid and its natural and semi-synthetic derivatives

Boropinic acid is a natural isopentenyloxycinnamic acid extracted from the aerial parts of Boronia pinnata Sm. (Rutaceae) with soybean 5-lipoxygenase inhibitory activity. In this paper the topical anti-inflammatory activity of boropinic acid and some of its natural and semi-synthetic derivatives was evaluated using the Croton oil ear test in mice as a model of acute inflammation. Some of the tested compounds (15, 17, 19, 20) revealed an effect comparable (ID₅₀ = 0.18 ÷ 0.72 μmol/cm²) to that of the reference drug indomethacin (ID₅₀ = 0.23 μmol/cm²), a non-steroidal anti-inflammatory drug.     

EPR and magnetic properties of [Mn(μ-Cl)2(bpy)]n: An unusual ferromagnetic interaction in a Mn(II) chloro-bridged polymer

The study of the magnetic properties and EPR spectra of the one-dimensional bis(μ-chloro) Mn(II) polymer [Mn(μ-Cl)₂(bpy)]_n (1) (bpy = 2,2′-bipyridine) is reported. Magnetic susceptibility measurements reveal a weak ferromagnetic interaction between the Mn(II) ions (J = +0.19 cm⁻¹). This is the first polymer of six-coordinated Mn(II) ions with a [Mn₂(μ-Cl)₂]²⁺ magnetic repeating unit, showing a ferromagnetic interaction. The coordination octahedra are elongated in the Cl?Cl direction and the elongation axes are parallel along the chain. Extended Hückel calculations show that the octahedra share an equatorial-apical edge, with the equatorial plane being parallel, and perpendicular to the Mn₂(μ-Cl)₂. Due to this disposition, the overlap through the chloride bridges is small, so the antiferromagnetic contribution must also be small and, therefore, a ferromagnetic behaviour is observed. The EPR spectra of a polycrystalline sample of 1 at different temperatures are also reported. A variation of the bandwidth with temperature is observed, which could be because this ferromagnetic interaction is weak: above 150 K the dipolar interaction is the predominant effect, while below 150 K the magnetic exchange interaction dominates.     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.     

A manganese(II) dichloro-bridged one-dimensional polymer: Structural, fluorescence and low-temperature magnetic study

One-dimensional organic/inorganic composite coordination polymer has been synthesised by the reaction of manganese(II) chloride with the chelating bidentate ligand, 1,10-phenanthroline (1,10-phen). X-ray single crystal analysis shows a doubly chloride bridged 1-D polymer, [Mn(μ-Cl)₂(phen)]_n (1), where manganese(II) ions possess octahedral environment. The complex is characterised by elemental analysis, different spectroscopic, electrochemical and low temperature magnetic susceptibility measurements. 1 exhibits strong fluorescence emission band at 410 nm and can serve as potential photoactive material as indicated from the characteristic fluorescence properties. Magnetic susceptibility measurements reveal a weak ferromagnetic interaction between the two high-spin Mn(II) ions of J = 0.017 cm⁻¹.     

Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide–tetrazolium coupling reactions for quantitative l-lactate analysis

In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2⁻⁶ U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25 U/μl), and NAD⁺ (2 μl, 1.5 × 10⁻⁵ M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10 mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5 mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications.     

Solid-phase extraction of tramadol from plasma and urine samples using a novel water-compatible molecularly imprinted polymer

In this study, a novel method is described for the determination of tramadol in biological fluids using molecularly imprinted solid-phase extraction (MISPE) as the sample clean-up technique combined with high-performance liquid chromatography (HPLC). The water-compatible molecularly imprinted polymers (MIPs) were prepared using methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross-linker, chloroform as porogen and tramadol as template molecule. The novel imprinted polymer was used as a solid-phase extraction (SPE) sorbent for the extraction of tramadol from human plasma and urine. Various parameters affecting the extraction efficiency of the polymer have been evaluated. The optimal conditions for the MIP cartridges were studied. The MIP selectivity was evaluated by checking several substances with similar molecular structures to that of tramadol. The limit of detection (LOD) and limit of quantification (LOQ) for tramadol in urine samples were 1.2 and 3.5 μg L⁻¹, respectively. These limits for tramadol in plasma samples were 3.0 and 8.5 μg L⁻¹, respectively. The recoveries for plasma and urine samples were higher than 91%.     

A lactate biosensor based on lactate dehydrogenase/nictotinamide adenine dinucleotide (oxidized form) immobilized on a conducting polymer/multiwall carbon nanotube composite film

An amperometric lactate biosensor was developed based on a conducting polymer, poly-5,2′-5′,2′′-terthiophene-3′-carboxylic acid (pTTCA), and multiwall carbon nanotube (MWNT) composite on a gold electrode. Lactate dehydrogenase (LDH) and the oxidized form of nicotinamide adenine dinucleotide (NAD⁺) were subsequently immobilized onto the pTTCA/MWNT composite film. The modified electrode was characterized by quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and electrochemical experiments. The detection signal was amplified by the pTTCA/MWNT assembly onto which a sufficient amount of enzyme was immobilized and stabilized by the covalent bond formation between the amine groups of enzyme and the carboxylic acid groups of the pTTCA/MWNT film. Experimental parameters affecting the sensor responses, such as applied potential, pH, and temperature, were assessed and optimized. Analytical performances and dynamic ranges of the sensor were determined, and the results showed that the sensitivity, stability, and reproducibility of the sensor improved significantly using pTTCA/MWNT composite film. The calibration plot was linear (r² = 0.9995) over the range of 5 to 90 μM. The sensitivity was approximately 0.0106 μA/μM, with a detection limit of 1 μM, based on a signal/noise ratio of 3. The applicability of the sensor for the analysis of l-lactate concentration in commercial milk and human serum samples was demonstrated successfully.     

Oxidative stress augments the secretion of atrial natriuretic peptide in isolated rat atria

Reactive oxygen species (ROS) play a role in cardiovascular diseases such as hypertension and heart failure. The objective of the present study was to investigate the role of endogenous ROS in atrial hemodynamics and ANP secretion in isolated perfused beating rat atria. Pyrogallol (a generator of superoxide anion, 0.1, 1 mM) or hydrogen peroxide (0.1, 1, 10, 30 mM) was perfused into atria paced at 1.2 Hz. Pyrogallol and hydrogen peroxide stimulated ANP secretion and concentration in a dose-dependent manner and dramatically decreased atrial contractility and translocation of extracellular fluid. The stimulatory effect of pyrogallol and hydrogen peroxide on ANP secretion was attenuated by the pretreatment with ascorbic acid (an antioxidant; 1 mM) and cariporide (an inhibitor of the Na⁺/H⁺ exchanger; 1 μM) but negative inotropic effect was not changed. U120 (a MAPK^erk pathway inhibitor; 10 μM) attenuated the stimulatory effect of hydrogen peroxide on ANP secretion. However, U120 augmented negative inotropic effect and stimulatory effect of ANP concentration induced by pyrogallol. Antioxidant such as N-acetyl cystein, gallate, propyl gallate, or ellagic acid did not cause any significant changes in atrial parameters. These results suggest that intracellular - formed ROS stimulates ANP secretion partly through activation of MAPK^erk pathway and Na⁺/H⁺ exchanger.     

Microwave driven wood liquefaction with glycols

Wood liquefaction with glycols using p-toluenesulfonic acid as the catalyst was carried out under microwave heating. With rapid heating and temperatures in the 190–210 °C range complete liquefaction was achieved in 7 min. Liquefaction efficiency was dependent on the choice of glycol. Simple glycols such as ethylene glycol and propylene glycol were more effective than higher analogues. The use of glycerol in mixtures with glycols showed a synergistic effect. Size exclusion chromatography was used to follow the gradual emergence of liquefaction products in solution as well as the recondensation products that start forming early in the reaction and precipitate from solution when molar masses of approx. 1 × 10⁴ g/mol are reached.     

A novel NADP-dependent dehydrogenase activity for 7α/β- and 11β-hydroxysteroids in human liver nuclei: A third 11β-hydroxysteroid dehydrogenase

Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP⁺ or NAD⁺, but not NADPH and NADH, as pyridine nucleotide cofactor with K_m values of 12 ± 2 and 390 ± 2 μM, compared to the K_m for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The K_m for corticosterone in the NADP⁺-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The K_cat values for nuclear activity with NADP⁺ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2.     

Preparation of high-capacity supports containing protein G immobilized to porous silica

This study examined the preparation of high-capacity silica supports containing immobilized protein G. A maximum content of 39 mg protein G/g silica was obtained when using 100 Å pore size silica, followed by 33 mg/g for 50 Å silica and 9.3-24 mg/g for 300-4000 Å silica. The surface coverage of protein G increased with pore size, with a maximum level of 0.037 μmol/m² being obtained for 4000 Å silica. These supports gave comparable apparent activities (i.e., 30-47% binding to rabbit immunoglobulin G [IgG]), with the highest binding capacities (71-77 mg IgG/g silica) being obtained for 50-100 Å silica.