Search

The present study described the development and evaluation of a new fully automated multiplex competitive immunoassay enabling the simultaneous detection of five water pollutants (okadaic acid (OA), 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine (atrazine), 2.4-dichlorophenoxyacetic acid (2,4-D), 2,4,6-trinitrotoluene (TNT), and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX)). The technology is taking advantage of an optical-clear pressure-sensitive adhesive on which biomolecules can be immobilized and that can be integrated within a classical 96-well format. The optimization of the microarray composition and cross-reaction was performed using an original approach where probe molecules (haptens) were conjugated to different carriers such as protein (bovine serum albumin or ovalbumin), amino-functionalized latex beads, or dextran polymer and arrayed at the surface of the adhesive. A total of 17 different probes were then arrayed together with controls on the adhesive surface and screened toward their specific reactivity and cross-reactivity. Once optimized, the complete setup was used for the detection of the five target molecules (less than 3 h for 96 samples). Limits of detection of 0.02, 0.01, 0.01, 100, and 0.02 μg L-1 were found for OA, atrazine, 2,4-D, TNT, and RDX, respectively. The proof of concept of the multiplex competitive detection (semiquantitative or qualitative) of the five pollutants was also demonstrated on 16 spiked samples. [ABSTRACT FROM AUTHOR]

A new technique is reported for the enhanced colon-metric detection of multiplexed hybridization onto porous membrane-based microarrays. This approach combines the use of horseradish peroxidase (HRP) as a label together with a chromogen substrate and a local production of the hydrogen peroxide required for substrate oxidation. This in situ production of coreagent is obtained using glucose oxidase (GOx) directly immobilized within the micro-array porous membrane mesh. The oxidation of glucose by the immobilized GOx produces hydrogen peroxide which itself enables the oxidation of TMB(3,3',5,5'-tetramethylbenzidine) by HRP and yields a blue precipitate on positive spots. Thanks to a coreagent overconcentration within the membrane, this design drastically surpasses the performances of the standard TMB/H2O2 kit used for peroxidase label detection. The obtained target limit of detection is then SO times lower (20 pM) than the one obtained with the standard kit approach, and the dynamic range expands at least one decade. Furthermore, the developed method was shown to compete well with the widely used alkaline phosphatase-BClP (5-bromo-4-chIoro-3-indolyl phosphate)/NBT (nitro blue tetrazolium chloride) readout while minimizing background signal. The method was finally successfully applied to the multiplex detection of single nucleotide polymorphisms (SNPs) in complex PCR samples. The background lowering was impacted here positively on the SNPs' detection by increasing the complementary noncomplementary signal ratio. [ABSTRACT FROM AUTHOR]

The effect of the ionic liquid 1-ethyl-3-methylimidazolium ethylsulfate ([Emim][EtSO4]) on the copper-catalyzed luminol chemiluminescence (CL) is reported. A drastic light emission enhancement is observed, related to a strong interaction between Cu2+ and the imidazolium ring. In these conditions, the CL reaction was able to produce light efficiently at pH as low as 6.5 (amplification factom Intensity+ILIntensity-IL = 2900). Interesting effects of [Emim][EtSO4] on the enzyme glucose oxidase activity were also evidenced, and advantages were taken from this enhancement to perform sensitive chemiluminescentglucose detection (LOD = 4μM) at pH 8.0. [ABSTRACT FROM AUTHOR]

Diazonium cation electrodeposition was investigated for the direct and electro-addressed immobilization of proteins. For the first time, this reaction was triggered directly onto diazonium-modified proteins. Screen-printed (SP) graphite electrode microarrays were studied as active support for this immobilization. A 10-microelectrode (eight working electrodes, 0.2 mm2 each; one reference; and one auxiliary) setup was used to study the addressing possibilities of the method. These electrode microarrays were shown to be able to covalently graft diazonium cations through electrochemical reduction. Cyclic voltammetry and X-ray photoelectron spectroscopy were used to characterize the electrochemical grafting onto our SP graphite surface and suggested that a diazonium monolayer was deposited. Rabbit and human immunoglobulins (lgGs) were then chemically coupled to an aniline derivative (4-carboxymethylaniline), followed by diazotation to form an aryl diazonium function available for the electrodeposition. These modified proteins were both successfully electro-addressed at the surface of the graphite electrodes without cross-talk or interference. The immuno-biochip obtained using this novel approach enabled the specific detection of anti-rabbit lgG antibodies with a detection limit of 50 fmol of protein. A promising strategy to immobilize markedly different biological entities was then presented, providing an excellent spatial specificity of the electro-addressing. [ABSTRACT FROM AUTHOR]

Peroxidase labeled streptavidin was immobilized onto the surface of bulk and clusterlike metal films at a distance controlled by a peptide chain with a length between 1.3 and 7.8 nm. Luminol chemiluminescence which occurred at peroxidase vicinity depends on the metal nanostructure. When peroxidase is attached on a bulklike film, chemiluminescence increases monotonously with the distance because of a decrease of the light emission quenching by metal. When peroxidase is attached on a clusterlike film, chemiluminescence undergoes a complex variation with the metal/catalyst distance evidencing a competition between the already mentioned quenching process and a nanostructure-induced catalysis enhancement. [ABSTRACT FROM AUTHOR]