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Vashist S.K.,Hsg Imit Institute For Mikro Und Informationstechnik | Vashist S.K.,Albert Ludwigs University of Freiburg | Schneider E.M.,University of Ulm | Lam E.,National Research Council Canada | And 2 more authors.
Scientific Reports | Year: 2014

An improved enzyme-linked immunosorbent (ELISA) assay using one-step antibody immobilization has been developed for the detection of human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The anti-HFA formed a stable complex with 3-aminopropyltriethoxysilane (APTES) by ionic and hydrophobic interactions. The complex adsorbed on microtiter plates exhibited a detection range of 4.9 pg mL-1 to 20 ng mL-1 HFA, with a limit of detection of 7 pg mL-1. Furthermore, an analytical sensitivity of 10 pg mL-1 was achieved, representing a 51-fold increase in sensitivity over the commercial sandwich ELISA kit. The results obtained for HFA spiked in diluted human whole blood and plasma showed the same precision as the commercial kit. When stored at 4°C in 0.1 M phosphate-buffered saline (PBS, pH 7.4), the anti-HFA bound microtiter plates displayed no significant decrease in their functional activity after two months. The new ELISA procedure was extended for the detection of C-reactive protein, human albumin and human lipocalin-2 with excellent analytical performance.

Zhou L.,Innovative Chromatography Group | Zhou L.,University College Cork | Reen F.J.,University College Cork | O'Gara F.,University College Cork | And 6 more authors.
Journal of Chromatography A | Year: 2012

Coated capillary electrophoresis equipped with a boron doped diamond (BDD) electrode was developed for analysis of chemically synthesised 2-heptyl-3-hydroxy-4-quinolone (HHQ), 2-heptyl-3-hydroxy-4-quinolone (PQS), and 2-methyl analogues. Detection was then extended to biological samples. PQS and its biological precursor, HHQ, are two key regulators of bacterial cooperative behaviour known as quorum sensing in the nosocomial pathogen Pseudomonas aeruginosa. The fused silica capillary was coated with a thin layer of poly (diallyldimethylammonium) chloride to reverse the electroosmosis, allowing fast migration of PQS and HHQ with improved selectivity. The four model compounds were baseline resolved using a 50mM H 3PO 4-Tris, pH 2.0 buffer with 20% (v/v) acetonitrile as buffer additive. With an injection time of 3s, the detection limits of four analytes ranging from 60 to 100nM (S/N=3) were observed when the BDD electrode was poised at +1.5V vs. 3M Ag/AgCl. As expected, no PQS or HHQ was detected from the supernatant of the P. aeruginosa (pqsA) mutant. A concentration of HHQ of 247μM was detected from the supernatant of the pqsH mutant, which catalyses the conversion of HHQ to PQS in the presence of molecular oxygen by monooxygenase. The separation and detection scheme was applicable to follow the conversion of HHQ to PQS in P. aeruginosa when entering the stationary phase of growth. The results obtained by coated capillary electrophoresis with BDD detection were validated and compared well with LC-MS data. © 2012 Elsevier B.V.

Shang F.,Innovative Chromatography Group | Shang F.,University College Cork | Muimhneachain E.O.,University College Cork | Jerry Reen F.,University College Cork | And 9 more authors.
Bioorganic and Medicinal Chemistry Letters | Year: 2014

Pseudomonas aeruginosa uses a hierarchical cell-cell communication system consisting of a number of regulatory elements to coordinate the expression of bacterial virulence genes. Sensitive detection of quorum sensing (QS) molecules has the potential for early identification of P. aeruginosa facilitating early medical intervention. A recently isolated cell-cell communication molecule, a thiazole termed IQS, can bypass the las QS system of P. aeruginosa under times of stress, activating a subset of QS-controlled genes. This compound offers a new target for pathogen detection and has been prepared in a one step protocol. A simple electrochemical strategy was employed for its sensitive detection using boron-doped diamond and glassy carbon electrodes by cyclic voltammetry and amperometry. © 2014 Elsevier Ltd. All rights reserved.

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