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AFM-based Nanoimmuno Assays

We have designed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. DNA nanoarrays were fabricated by means of AFM based nanografting and antibodies were then immobilized using DNA-directed immobilization of DNA-protein conjugates. We tested the efficiency of our assay in standard solution and in cellular lysate and we compared it with conventional ELISA assay. Ambrosetti et al. ACS Omega 2, 2618 (2017)

 

      

Early detection of cancer plays a crucial role in disease prognosis. It requires the recognition and quantification of low amounts of specific molecular biomarkers, either free or transported inside nanovesicles, through the development of novel sensitive diagnostic technologies. In this context, we have developed a nanoarray platform for the noninvasive quantification of cancer biomarkers circulating in the bloodstream. The assay is based on molecular manipulation to create functional spots of surface-immobilized binders and differential topography measurements. It is label-free and requires just a single binder per antigen, and when it is implemented with fluorescence labeling/readout, it can be used for epitope mapping. As a benchmark, we focused on the plasma release of Her2 extracellular domain (ECD), a proposed biomarker for the progression of Her2-positive tumors and response to anticancer therapies. By employing robust, easily engineered camelid nanobodies as binders, we measured ECD-Her2 concentrations in the range of the actual clinical cutoff value for Her2-positive breast cancer. The specificity for Her2 detection was preserved when it was measured in parallel with other potential biomarkers, demonstrating a forthcoming implementation of this approach for multiplexing analysis. Prospectively, this nanorarray platform may be customized to allow for the detection of promising new classes of circulating biomarkers, such as exosomes and microvesicles.

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Quantification of Circulating Cancer Biomarkers via Sensitive Topographic Measurements on Single Binder Nanoarrays
E. Ambrosetti, P. Paoletti, A. Bosco, P. Parisse, D. Scaini, E. Tagliabue, A. de Marco, L. Casalis
ACS Omega 2, 2618 (2017)

Last Updated on Monday, 12 February 2018 15:27