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Aptamer-functionalized nanopore for the detection of closely-related proteins

  • Reynaud, Lucile
Publication Date
Nov 24, 2020
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The specific detection of proteins is a major challenge in many biotechnological fields such as biomedical diagnostics or fundamental understanding complex protein interactions. Solid-state nanopores are microfabricated nanoscale apertures in thin dielectric membranes that have raised attention as label-free single-molecule biosensors with high sensitivity. A voltage is applied across the membrane, which drives biomolecules through the nanopore. When the analyte crosses the nanopore, it causes a current blockage for a certain time that provides a specific electrical signature for each molecule of interest. During this thesis, a cleanroom process flow has been developed for the fabrication of nanopore chips as well as the drilling of 15 nm diameter nanopore in a 20 nm thick silicon nitride membrane using a Transmission Electron Microscope. The experimental bench for nanopore sensing has been built up and tested. α-thrombin, ɣ-thrombin (5 nm diameter globular proteins) and prothrombin (5 nm x 9 nm oblate spheroid) are three closely-related proteins involved in blood coagulation cascade. They have been sensed using a bare nanopore down to 1 nM concentration, and prothrombin could be discriminated thanks to its bigger size. In order to discriminate α-thrombin from ɣ-thrombin, which have similar sizes, the nanopore’s surface has been chemically functionalized with aptamers. Aptamers are short single stranded DNA sequences selected for their affinity towards a specific biomolecule. We used an aptamer specifically recognizing α-thrombin. We demonstrated that the nanopore functionalization was successful thanks to a measured diameter reduction of the nanopore. Moreover, α-thrombin and ɣ-thrombin present different electrostatic interactions with the functionalization surface of the nanopore, hence a different dwell-time signature in the pore and could be discriminated. Aptamer-functionalized nanopores provide promising and versatile biosensing performances.

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