Ram Abuknesha

Integrated optical surface plasmon resonance immunoprobe for simazine detection

This paper presents the detailed design and characterisation of a regenerable integrated optical surface plasmon resonance immunoprobe as a detector for the triazine herbicide simazine. A sensor design theoretically optimised for use in the aqueous environment is presented and its fabrication described. Experimental results on the sensitivity to changes in bulk refractive index of the analyte and on non-specific binding of ovalbumin are presented. Binding inhibition immunoassays were conducted for simazine and the lower limit of detection determined to be 0.16 microgram/l using anti-simazine IgG antibodies and 0.11 microgram/l using anti-simazine Fab fragments. A sample test cycle of 20 min was established.

Immunofluorescence sensor for water analysis

We demonstrated a bulk optical fluorescence based immunosensor capable of multianalyte water analysis. Calibration curves obtained for 2.3-dichlorophenoxyacetic acid (2,4-D) and simazine had detection limits of 0.035µg/l and 0.026µg/l respectively. The sensor is reusable due to its regenerability and cost effective due to the use of components customary in the trade. Ways to further enhance device sensitivity by means of a high index film deposited on the sensor surface or by employing an integrated optical waveguide as transducer are presented. A concept for the detection of a varying range of analytes on the same transducer is discussed.

Antibody-antigen-adjuvant conjugates enable co-delivery of antigen and adjuvant to dendritic cells in cis but only have partial targeting specificity

Antibody-antigen conjugates, which promote antigen-presentation by dendritic cells (DC) by means of targeted delivery of antigen to particular DC subsets, represent a powerful vaccination approach. To ensure immunity rather than tolerance induction the co-administration of a suitable adjuvant is paramount. However, co-administration of unlinked adjuvant cannot ensure that all cells targeted by the antibody conjugates are appropriately activated. Furthermore, antigen-presenting cells (APC) that do not present the desired antigen are equally strongly activated and could prime undesired responses against self-antigens. We, therefore, were interested in exploring targeted co-delivery of antigen and adjuvant in cis in form of antibody-antigen-adjuvant conjugates for the induction of anti-tumour immunity. In this study, we report on the assembly and characterization of conjugates consisting of DEC205-specific antibody, the model antigen ovalbumin (OVA) and CpG oligodeoxynucleotides (ODN). We show that such conjugates are more potent at inducing cytotoxic T lymphocyte (CTL) responses than control conjugates mixed with soluble CpG. However, our study also reveals that the nucleic acid moiety of such antibody-antigen-adjuvant conjugates alters their binding and uptake and allows delivery of the antigen and the adjuvant to cells partially independently of DEC205. Nevertheless, antibody-antigen-adjuvant conjugates are superior to antibody-free antigen-adjuvant conjugates in priming CTL responses and efficiently induce anti-tumour immunity in the murine B16 pseudo-metastasis model. A better understanding of the role of the antibody moiety is required to inform future conjugate vaccination strategies for efficient induction of anti-tumour responses.

Biosensors for unattended, cost-effective and continuous monitoring of environmental pollution: Automated Water Analyser Computer Supported System (AWACSS) and River Analyser (RIANA)

This work describes our recent progress and achievements in the field of fully automated biosensors (Automated Water Analyser Computer Supported System (AWACSS) and River Analyser (RIANA)) for unattended, cost-effective and continuous monitoring of environmental pollution. We report on ultra-sensitive immunoassays for the hormones progesterone, testosterone and estrone and the pesticides propanil and isoproturon as examples of the outstanding progress made on biosensors in the field of environmental monitoring and water analysis. Most of the bio-active organic pollutants (estrone, progesterone, propanil and isoproturon) were detected at levels as low as 1.0 pg mL−1 or even below. In fact, the reported limits of detection (LOD) were between 0.2 and 6.0 pg mL−1. For the first time, commercially available derivatives and antibodies were incorporated into immunoassays (progesterone and testosterone) for fully automated biosensors. To verify the assay performance for quantifying testosterone, progesterone, and isoproturon in real-world samples using our immunosensors, we spiked river and drinking water at six different levels from 0.9 pg mL−1 to 90 ng mL−1. Nearly all recovery rates could be obtained between 70 and 120% as the AOAC International recommends it chiefly for water analysis.

Waveguide immunofluorescence sensor for water pollution analysis

A regenerable channel waveguide fluorescence sensor for environmental monitoring is reported. The sensor has been characterized as a detector of the pesticide 2,4 dichlorophenoxyacetic acid. A binding inhibition assay, using fluorescent Cy5.5 dye-labeled antibodies, was monitored at the modified surface of the glass waveguide to detect the target analyte. Three calibration curves were determined and averaged. The averaged calibration curve has a mid-point of 0.68 ppb and a calculated detection limit of 0.28 ppb. Incorporation of a 20 nm thick tantalum pentoxide film at the waveguide surface enhanced the peak fluorescence signal by a factor of approximately 6 compared with an uncoated sensor.

Integrated optical immunofluorescence multisensor for river pollution

A 32-analyte integrated optical immunofluorescence multisensor system has been realized and tested for the first time. The sensor system is based upon bio/immuno-chemistry at the waveguide surface and fluoroimmunoassay in the evanescent fields of the optcal waeguides, to enable rapid, simultaneous and high-sensitivity fluorescence detection of up to 32 pollutants in water, and automatic regeneration for immediate reuse. The system has been demonstrated for estrone and a detection limit of 13 ng/L has been achieved.

River Analyser — Multiresidue Immunoanalytical Monitoring Tools

Immunoanalytical techniques find growing acceptance in the field of environmental monitoring (Van Emon and Gerlach 1995). Although immunoassays have many attractive features, the lack of multiresidue approaches is a serious obstacle in environmental monitoring. In many environmental situations the simultaneous determination of more than one analyte is required. The situation is further complicated by the fact, that the analyte panels, which are to be detected, may vary with location and season. The RIANA project (RIver ANAlyser) is focused on immunoanalytical tools, that allow to detect multiple analytes in a single run. The application areas foreseen are online monitoring - e.g. at pumping sites or transportable devices for field use. In this paper we describe the concept and give first results.