Acetylcholinesterase amperometric detection system based on a cobalt(II) tetraphenylporphyrin-modified electrode

An acetylcholinesterase (AChE) activity detection system was fabricated based on the electrocatalysis of cobalt(II) tetraphenylporphyrin of the electrooxidation of thiocholine chloride, which is the product of the hydrolysis of acetylthiocholine chloride by AChE. A simple modified method was used to form the base electrode. AChE was cross-linked on the base electrode by glutaraldehyde. The optimum working conditions are discussed and the characteristics of the detection system are evaluated.

Kinetic Study of Prolidase Activity in Erythrocytes against Different Substrates Using Capillary Electrophoresis with Electrochemiluminescence Detection

Capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection was used to explore the kinetics ofthe enzymatic reaction. The different effects ofreaction conditions including the concentration of Mn2l, incubation temperature and pH on PFOlidase (PLD, EC 3.4.13.9) activity in erythrocyte lysates against three different substrates, Gly-Pro, Val-Pro and Leu-Pro were investigated. Also, the effects of colchicine which can prevent or delay cancer ofliver on the PLD activity were studied.

Characterization of prolidase activity using capillary electrophoresis with tris(2,2'-bipyridyl)ruthenium(II) electrochemiluminescence detection and application to evaluate collagen degradation in diabetes mellitus

A new method for prolidase (PLD, EC 3.4.13.9) activity assay was developed based on the determination of proline produced from enzymatic reaction through capillary electrophoresis (CE) with tris(2,2'-bipyridyl)ruthenium(11) [Ru(bpy)(3)(2+)] electrochemiluminescence detection (ECL). A detection limit of 12.2 fmol (S/N = 3) for proline, corresponding to 1.22 x 10(-8) units of prolidase catalyzing for 1 min was achieved. PLD activity determined by CE-ECL method was in agreement with that obtained from the classical Chinard's one. CE-ECL showed its powerful resolving ability and selectivity as no sample pretreatmentwas needed and no interference existed. The clinical utility of this method was successfully demonstrated by its application to assay PLD activity in the serum of diabetic patients in order to evaluate collagen degradation in diabetes mellitus (DM). The results indicated that enhanced collagen degradation occurred in DM.

Kinetic study of paracetamol on prolidase activity in erythrocytes by capillary electrophoresis with Ru(bpy)(3)(2+) electrochemiluminescence detection

We explored the CE with Ru(bpy)(3)(2+) electrochemiluminescence detection for the kinetic study of drug-enzyme interaction. Effects of four nonsteroidal anti - inflammatory drugs including aspirin, paracetamol, sodium salicylate and phenacetin on prolidase (PLD) activity in erythrocytes were investigated. Aspirin enhanced PLD activity whereas the other three had inhibiting effects. This may reveal their different effects on the collagen biosynthesis and catabolism that influence tumor invasiveness. Kinetic study of paracetamol on PLD showed that the value of Michaelis constant Km for PLD was 1.23 mM. The mechanism of PLD inhibition by paracetamol is noncompetitive inhibition, and the inhibitor constant K-i value obtained in our research was 9.73 x 10(3) mu g/L.

Detection of microcystins in environmental samples using surface plasmon resonance biosensor

An indirect inhibitive surface plasmon resonance (SPR) immunoassay was developed for the microcystins (MCs) detection. The bioconjugate of MC-LR and bovine serum albumin (BSA) was immobilized on a CM5 sensor chip. A serial premixture of MC-LR standards (or samples) and monoclonal antibody (mAb) were injected over the functional sensor surface, and the subsequent specific immunoreaction was monitored on the BIAcore 3000 biosensor and generated a signal with an increasing intensity in response to the decreasing MCs concentration. The developed SPR immunoassay has a wide quantitative range in 1-100 mu g L-1. Although not as sensitive as conventional enzyme-linked immunosorbent assay (ELISA), the SPR biosensor offered unique advantages: (I) the sensor chip could be reusable without any significant loss in its binding activity after 50 assay-regeneration cycles, (2) one single assay could be accomplished in 50 min (including 30-min preincubation and 20-min BIAcore analysis), and (3) this method did not require multiple steps. The SPR biosensor was also used to detect MCs in environmental samples, and the results compared well with those obtained by ELISA. We conclude that the SPR biosensor offers outstanding advantages for the MCs detection and may be further developed as a field-portable sensor for real-time monitoring of MCs on site in the near future. (C) 2009 Published by Elsevier B.V.

Antimicrobial activity-specific to Gram-negative bacteria and immune modulation-mediated NF-kappa B and Sp1 of a medaka beta-defensin

Defensins are a group of cationic antimicrobial peptides which play an important role in the innate immune system by exerting their antimicrobial activity against pathogens. In this study, we cloned a novel beta-defensin cDNA from medaka (Oryzias latipes) by rapid amplification of cDNA ends (RACE) technique. The full-length cDNA consists of 480 bp, and the open reading frame (CRF) of 189 bp encodes a polypeptide of 63 amino acids (aa) with a predicted molecular weight of 7.44 kDa. Its genomic organization was analyzed, and Southern blot detection confirmed that only one copy of beta-defensin exists in the medaka HNI strain. RT-PCR, Western blot and immunohistochemistry detections showed that the beta-defensin transcript and protein could be detected in eyes, liver, kidney, blood, spleen and gill, and obviously prevalent expression was found in eyes. Antimicrobial activity of the medaka beta-defensin was evaluated, and the antibacterial activity-specific to Gram-negative bacteria was revealed. Furthermore, the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was demonstrated to be able to induce about 13-fol up-regulation of the beta-defensin within first 12 h. In addition, promoter and promoter mutagenesis analysis were performed in the medaka beta-defensin. A proximal 100 base pair(bp) sequence (+26 to -73)and the next 1700 bp sequence (-73 to -1755) were demonstrated to be responsible for the basal promoter activity and for the transcription regulation. Three nuclear factor kappa B (NF-kappa B) cis-elements and a Sp1 cis-element were revealed by mutagenesis analysis to exist in the 5' flanking sequence, and they were confirmed to be responsible for the up-regulation of medaka beta-defensin stimulated by LPS. And, the Sp1 cis-element was further revealed to be related to the basal promoter activity, and transcriptional factor II D (TFIID) was found to be in charge of the gene transcription initiation. All the obtained data suggested that the novel medaka beta-defensin should have antimicrobial activity-specific to Gram-negative bacteria, and the antibacterial immune function should be modulated by NF-kappa B and Sp1. (C) 2008 Elsevier Ltd. All rights reserved.

Detection and quantitative analysis of polychlorinated biphenyls in tilapia from Hawaiian waters

The purpose of this study was to investigate polychlorinated biphenyls (PCBs) contamination in tilapia (Oreochromis mossambicus) collected from the Manna stream and Ala Wai Canal of O'ahu, an island of the geographically isolated Hawaiian archipelago. Our results show that the average concentrations of PCBs varied from 51.90 to 89.42 ng g(-1) lipid weight for the sampling sites. Relative toxic potencies (RTPs) and toxic equivalencies (TEQs) were determined to be 20.38-40.60 ng TCDD g(-1) lipid weight and 2.89-4.17 ng TEQ g(-1) lipid weight by 7-ethoxy-resorufin-O-deethylase (EROD) activity analysis and calculation of PCB concentrations based on toxic equivalency factors (TEFs), respectively. Penta-chlorinated congeners were found to be predominant, which revealed that Aroclor 1254 was a possible major source of PCBs in our fish samples. PCB 118, an indicator PCBs, constituted more than 55% and 30% of the total PCBs and TEQs, respectively. In addition, PCB 118 was found to have a linear correlation to the total PCBs (R = 0.975) and TEQs (R = 0.782). Detection of concentrated PCBs in Hawaiian waters suggests a potentially adverse impact of this pollutant on human health, as well as ecological systems, and suggests the necessity of environmental monitoring and hazard assessment of PCBs within the Hawaiian Islands. (c) 2008 Published by Elsevier Ltd.

Assessment of estrogenic activity of leachate from automobile tires with two in vitro bioassays

In this study, a combination of enzyme-linked receptor assay (ELRA) and yeast estrogen screen (YES) assay was firstly applied to determine whether automobile tires immersed in fresh water can leach chemicals, which display estrogenic activity. We optimized ELRA substituting the chromogene substrate by a luminescent one, and found that luminescent ELBA was more sensitive to 17 beta-estradiol (17 beta-E2) with a detection limit of 0.016 mu g/l, compared to 0.088 mu g/l in the chromogene version. In ELRA, all tire leachates obviously showed estrogenic activity, which was increased with duration of immersion. Moreover, the leachate from hackled tires showed more potent estrogenicity than that from the whole ones. In comparison to ELRA, no detectable estrogenic activity was found in all tire leachates with YES assay. The results from YES assay further evidenced that antiestrogenic compounds can be leached from tires. As tire leachates contain estrogenic compounds, they could be important pollution sources, potentially harmful to wildlife and human health. Thus, use of shredded tires as road fill or in landfill sites should arouse our attention.

Detection of extracellular phosphatases in natural spring phytoplankton of a shallow eutrophic lake (Donghu, China)

The species-specific production of extracellular phosphatases in phytoplankton of a subtropical polymictic take was investigated from March to May 2004. Phosphatase activity was detected directly at the site of enzyme action using the enzyme-labelled fluorescence (ELF) technique. Size fractionation of bulk phosphatase activity (PA), concentrations of soluble reactive phosphorus (SRP), chlorophyll a, and phytoplankton composition were determined in parallel. Phosphatase-positive cells were present in every phytoplankton sample; labelled cells were detected in 33 algal taxa, including many chlorophytes, dinoflagellates and some diatoms, but never among cyanobacteria. We recorded an unusual dinoflagellate bloom (Peridiniopsis sp.), of which similar to 25% of the cells were phosphatase-positive. Several populations were partly phosphatase-positive whenever present, while some other species never showed any activity. The production of extracellular phosphatases was not primarily regulated by ambient P concentrations; algae produced these enzymes even if SRP concentrations were high. Moreover, heterotrophic nanoflagellates most probably contributed to the pool of particle-bound PA in some samples.

Electrochemical characteristics of mediated laccase-catalysis and electrochemical detection of environmental pollutants

Laccase has been immobilized on the carbon nanotubes modified glassy carbon electrode surface by adsorption. As-prepared laccase retains good electrocatalytic activity to oxygen reduction by using 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as the mediator. It can be used as a biosensor for the determination of catechol with broad linear range.

Fe3O4 magnetic nanoparticles as peroxidase mimetics and their applications in H2O2 and glucose detection

Artificial enzyme mimetics are a current research interest because natural enzymes bear some serious disadvantages, such as their catalytic activity can be easily inhibited and they can be digested by proteases. A very recently study reported by Yan et al. has proven that Fe3O4 magnetic nanoparticles (MNPs) exhibit an intrinsic enzyme mimetic activity similar to that found in natural peroxidases, though MNPs are usually thought to be biological and chemical inert (Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.; Feng, J.; Yang, D. L.; Perrett, S.; Yan, X. Y. Nat. Nanotechnol. 2007, 2, 577-583).

G-quadruplex-based DNAzyme for facile colorimetric detection of thrombin

Thrombin-binding aptamer is found to bind hemin to form a catalytic complex whose activity is significantly promoted by the addition of thrombin, which enables the colorimetric detection of thrombin with high specificity and sensitivity in a facile way.

Chemiluminescence thrombin aptasensor using high-activity DNAzyme as catalytic label

With a designed high-activity DNAzyme as the catalytic label, an ultrasensitive chemiluminescence thrombin aptasensor is developed, enabling a 10- to 100-fold improvement in the detection sensitivity as compared with previous counterparts.

Electrochemical Detection of Hydrazine Based on Electrospun Palladium Nanoparticle/Carbon Nanofibers

In this work, we developed an electrochemical method for the detection of hydrazine based oil palladium nanoparticle/carbon nanofibers (Pd/CNFs). Pd/CNFs were prepared by electrospinning technique and subsequent thermal treatments. The electrocatalytic behaviors of Pd/CNFs modified glassy carbon electrode (Pd/CNF-GCE) for hydrazine oxidation were evaluated by cyclic voltammetry (CV), an obvious and well-defined oxidation peak appeared at -0.32 V (vs. Ag/AgCl). The mechanism of the oxidation of hydrazine at Pd/CNF-GCE was also studied, which demonstrated an irreversible diffusion-controlled electrode process and a four-electron transfer involved in the overall reaction. Furthermore, the wide linear range, low detection limit, good reproducibility and excellent storage stability were obtained utilizing differential pulse voltammetry (DPV).

Multifunctional G-Quadruplex Aptamers and Their Application to Protein Detection

Two significant G-quadruplex aptamers named AGRO100 and T30695 are identified as multi functional aptamers that can bind the protein ligands nucleolin or HIV-1 integrase and hemin. Besides their strong binding to target proteins, both AGRO100 and T30695 exhibit high hemin-binding affinities comparable to that of the known aptamer (termed PS2M) selected by the in vitro evolution process. Most importantly, their corresponding hemin-DNA complexes reveal excellent peroxidase-like activities. higher than that of the reported hemin-PS2M DNAzyme. This enables these multifunctional aptamers to be applied to the sensitive detection of proteins. which is demonstrated by applying AGRO100 to the chemiluminescence detection of nucleolin expressed at the surface of HeLa cells. Based on the specific AGRO100-nucleolin interaction, the surface-expressed nucleolin of HeLa cells is labeled in situ with the hemin-AGRO100 DNAzyme, and then determined in the luminol-H2O2 system.