Nanotechnology Applications in Quantitative Neuroscience: Proteomic Analysis of Malignant Gliomas

Abstract (English) The current limit of knowledge advancement in proteomic analysis of gliomas, the most common primary malignant brain tumors, is related to the high sensitivity required to detect specific biomarkers within few cells volumes. To address this problem we developed a quantitative approach to eventually enable precise, high throughput and low cost analysis of glial cells with potential capability of real-time pathological screening and subtyping of brain tumors. A device consisting in micro-fabricated wells capable to isolate and host living astrocytes was designed and functionalized. Then for the fabrication of a nanobiosensor, able to detect in small volumes the presence of specific biomarkers, ideally for multiplexing assays and meant to fit within the small dimensions of this microdevice, an approach consisting in DNA-directed-immobilization (DDI) of biotinylated antibodies (Abs) on a single stranded DNA (ssDNA) nanoarray, produced by Atomic Force Microscopy (AFM) nanografting, was carefully optimized. The proof of concept was realized with Abs specific for Glial Fibrillary Acidic Protein (GFAP), a biomarker which belongs to the family of intermediate filaments and is crucial in cell's differentiation, within a platform ready for parallelization. Nanosized patches of thiol modified ssDNA were prepared by AFM-based nanografting inside a matrix of self assembled monolayers (SAM) of alkanethiol-modified gold surfaces. Subsequently a complementary DNA strand (cDNA) conjugated to streptavidin (STV) was allowed to covalently bind to the patch by sequence specific DNA hybridization. Finally the biotin binding sites of STV were exploited to immobilize biotinylated monoclonal GFAP Abs (already in use for ELISA assays) on the top of those nanopatches. The efficiency of those nano-immuno arrays was tested by successfully obtaining the immobilization of purified recombinant GFAP protein, down to a concentration of 4 nM, firstly in standard PBS then in multicells' lysate obtained from U87 glial cultures. The immobilization was detected by means of AFM measuring step by step the increases in the height of the patches and excluding modification of the roughness of both the SAM and the nanopatches after incubation with the cells' lysate through a signal to noise ratio analysis. Titration curves for a comparison of sensitivity between this technique and the conventional ELISA assays are provided, they indeed confirm that the sensitivity of our nanosensors is at least that of ELISA, with the advantage of the scalability of the device.