Nanoscaled Architectures for highly Sensitive Biosensing of Small molecules
Final Report Abstract
Rapid and accurate detection of undesirable and toxic substances in human environment has become a major concern in our modern society. Consequently, biosensors are now becoming part of daily routine. These analytical devices incorporate a biological material intimately associated with a physicochemical transducer and enable detection of a target molecule, specifically and rapidly, even in trace amounts and even in demanding milieus. Biosensors elaboration is based on the controlled immobilization of a molecular receptor, an antibody in the case of immunosensors, on a transducer surface. Many efforts have been made to improve the sensitivity of these immunosensors, especially for the detection of targets of low molecular weight (haptens). The strategy we adopted for this project relied on three main tasks to face the actual challenges of hapten immunosensors and develop a new generation of ultra-sensitive and specific immunosensor platforms. The first task was obtaining the best (high quality) antibodies relying on modern biomolecular methods. Highly affine monoclonal antibodies (mAb) against benzo[a]pyrene (B[a]P) and microcystin-LR (MC-LR) were produced and first time new mAb was generated for diclofenac (DCF) by the German partner (TUM) and made available to the French project partners (Univ Paris). All antibodies are highly affine (KD-values in the nanomolar-picomolar range) and take a top position on the international scale. The second task was to optimize immobilization of antibodies by surface nanostructuration using three original strategies at the nanoscale, i.e., gold nanoparticles, polyoxometalates or layered silicates, to generate layers with improved accessibility and sensor sensitivity. Gold nanoparticles, prepared by the French partner, were biofunctionalized with the antibodies, but did not lead to detection enhancement of the performed automated flow-through chemiluminescence immunoassay, as expected. Whereas, polyoxometalate (POM) was successfully used to achieve surface nanostructuration onto planar gold surfaces through covalent reaction with mercaptoundecanoic acid self-assembled monolayer adsorbed on gold. The detection of B[a]P in the indirect competitive immunoassay format was carried out and the binding to the immunoprobe monitored with a quartz crystal microbalance with dissipation measurement (QCM-D). Further, two forms of layered silicates, ilerite and magadite, were synthesized, intercalated with a bulky anion, and their surfaces functionalized with two silanes. In task ‘3’, developments of tasks ‘1’ and ‘2’ should be combined to develop different assays and test the performance. In detail, the performance of the POM-structured QCM-D biosensor displayed enhanced accessibility on the surface and improved analytical performance. The microarray chip-based assay, using diaminopolyethyleneglycol-modified glass surface and covalently bond target analytes showed reproducible results in the ng/L-range for DCF and MC-LR. For DCF, concentrations at the low ppt-range were measured in real river water and lake water samples, and higher values of 2.9 and 2.1 μg/L were found in wastewater influents and effluents, respectively. These results could be confirmed by solid phase extraction combined with LC-MS reference method.
Publications
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Bifunctional Polyoxometalates for planar gold surfaces nanostructuration and protein immobilization. Journal of Physical Chemistry C, 2012, 116, 13217−13224
Mercier, D.; Boujday, S.; Annabi, C.; Villanneau, R.; Pradier, C.-M.; Proust, A.
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Optimizing the immobilization of gold nanoparticles on functionalized silicon surfaces: amine vs thiol terminated silane. Gold Bulletin, 2013, 46(4), 335–341
Ben Haddada, M., Blanchard, J., Casale, S., Krafft, J-M., Vallée, A, and Boujday, S.
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Layer-by-layer generation of PEG-based regenerableimmunosensing surfaces for small-sized analytes. Biosensors & Bioelectronics, 2015, 67, 334–341
Huebner, M.; Ben Haddada, M., Méthivier, C.; Niessner, R.; Knopp, D.; Boujday, S.
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Polyoxometalate nanostructured gold surfaces for sensitive biosensing of benzo[a]pyrene. Sensors and Actuators B, 2015, 209, 770–774
Mercier, D.; Ben Haddada M., Huebner M., Knopp D., Niessner R., Salmain, M.; Proust, A.; Boujday, S.
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Rapid analysis of diclofenac in fresh water and wastewater by a monoclonal antibody-based highly sensitive ELISA, Analytical and Bioanalytical Chemistry, 2015, 407, 8873-8882
Huebner, M.; Niessner, R.; Weber, E.; Boujday, S.; Knopp, D.
