Ioana Surugiu

An enzyme-linked molecularly imprinted sorbent assay

Based on a molecularly imprinted polymer, a competitive binding assay analogous to competitive enzyme immunoassay has been developed. The assay is specific for the herbicide 2,4-dichlorophenoxyacetic acid and uses, for the first time, an enzyme-labelled conjugate as a tracer. The label tobacco peroxidase allowed for colorimetric and chemiluminescence detection. The molecularly imprinted polymer was synthesised in the form of microspheres by precipitation polymerisation. The polymer efficiently and selectively bound the analyte in aqueous solution. Calibration curves were obtained corresponding to analyte concentrations ranging from 40–600 μg mL−1 for the colorimetric assay, and from 1–200 μg mL−1 for chemiluminescence assay.

Flow injection analysis of mercury(II) based on enzyme inhibition and thermometric detection

An enzymatic procedure for the determination of mercury(II) is described, based on inhibition of invertase using glucose oxidase and catalase co-immobilised on controlled-pore glass (CPG) coupled to a thermometric continuous-flow sensor system to follow the invertase activity. A small amount of invertase (0.66 U ml−1) was incubated for a short time in sucrose solution and 20 μl of the mixture were injected into the enzyme thermistor system to give a temperature change corresponding to 100% enzyme activity. Addition of a mercury(II) sample to the mixture caused a decrease in the invertase activity, that allowed the determination of mercury(II) concentrations in the 5–80 ppb range with RSD ⩽ 0.74%. The analysis time was 2–6 min including incubation. The main advantages of this thermometric biosensor assay are as follows: simplicity, with no need for regeneration due to the use of a cheap, soluble sensing enzyme; robustness, with excellent reproducibility and repeatability; and long operational and storage stability of the enzymes involved in the detection system.

Functionalized surfaces for optical biosensors: applications to in vitro pesticide residual analysis.

Functionalized biosensing surfaces were developed for chemiluminescent immunoassay of pesticides. Two approaches to construct functionalized surfaces were tested: (i) pesticide is immobilized to the surface and interacts with a labeled antibody; (ii) antibody is immobilized and interacts with a labeled pesticide. As labels alkaline phosphatase and peroxidase were used with their corresponding substrates CSPD and luminol, respectively. Light produced by chemiluminescent substrate was detected by a thermoelectrically cooled CCD camera or a photomultiplier. The best detection limit 0.00001 ng/ml was obtained using antibodies immobilized to dextran-enhanced surface. Completely renewable surface was obtained using reversible lectin-monosaccharide interaction, one surface was used for 200 analyses without any loss of binding capacity. Most favorable stability and cost per analysis was achieved with molecularly imprinted polymer (MIP) instead of antibody. The functionalized biosensing surfaces were prepared to detect 2,4-dichlorophenoxyacetic (2,4-D) acid as a model pesticide. The developed concepts are, however, generally applicable to other pesticides and to other optical formats, e.g. optical fiber.© 2001 Kluwer Academic Publishers

Immuno-arrays for multianalyte analysis of chlorotriazines

In this paper, a novel strategy for multicomponent analysis of two classes of pesticides such as triazines (atrazine and simazine) and phenoxyalkanoic acids (2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 4-chlorophenoxyacetic acid (CPOAc), phenoxyacetic acid (POAc)) employing immuno-arrays is demonstrated. The approach is based on cross-reactive arrays of specific antibody pairs coupled to chemometric pattern recognition. The monoclonal antibody pairs employed in this work (atrazine-simazine and 2,4-D) are specific towards a set of analytes and preclude a particular set of others present in the sample matrix. Antibody pairs of atrazine, simazine, and 2,4-D are used to discriminate and quantify analyte of interest. Atrazine was quantified in presence of trace concentration of simazine and that of 2,4-D. The combinatorial cross-reactivity of antibody pairs towards simazine, atrazine and 2,4-D is used to distinguish among different classes of analytes and their influence on the signal suppression in immuno-techniques. These sensors exclude recognition by carbamates such as carbaryl and carbofuran.

Dextran-modified surface for highly sensitive chemiluminescent ELISA

A highly sensitive chemiluminescent ELISA for 2,4-dichlorophenoxyacetic acid (2,4-D), based on disposable thin-glass capillaries, was developed. The amino-silanised glass capillaries were coated by covalently attaching activated carboxymethyl (CM) dextran of different sizes, to which monoclonal antibodies were coupled. A competitive immunoassay was performed using horseradish peroxidase (HRP)-labelled 2,4-D and a commercially available chemiluminescent substrate. The bound fraction of the 2,4-D-HRP conjugate was quantified by measuring the intensity of the emitted light using a photomultiplier tube detector (PMT). The proposed method resulted in highly improved sensitivity with an IC50 value of 0.002 ng ml−1 for capillaries coated with high molecular weight dextran.