Ultrasensitive and Selective Protein Recognition with Nanobody‐Functionalized Synthetic Nanopores. Issue 33 (3rd July 2021)

• Record Type: Journal Article
• Authors: Duznovic, Ivana; Gräwe, Alexander; Weber, Wadim; Müller, Lena K.; Ali, Mubarak; Ensinger, Wolfgang; Tietze, Alesia; Stein, Viktor
• Is Part Of: Small. Volume 17:Issue 33(2021)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2021-07-03
• Subjects: biosensors -- current rectification -- molecular diagnostics -- nanobodies -- nanofluidic devices -- synthetic nanopores -- track‐etched membrane; Nanotechnology -- Periodicals; Nanoparticles -- Periodicals; Microtechnology -- Periodicals; 620.5
• DOI: 10.1002/smll.202101066
• Languages: English
• ISSNs: 1613-6810

Abstract

Abstract: The development of flexible and reconfigurable sensors that can be readily tailored toward different molecular analytes constitutes a key goal and formidable challenge in biosensing. In this regard, synthetic nanopores have emerged as potent physical transducers to convert molecular interactions into electrical signals. Yet, systematic strategies to functionalize their surfaces with receptor proteins for the selective detection of molecular analytes remain scarce. Addressing these limitations, a general strategy is presented to immobilize nanobodies in a directional fashion onto the surface of track‐etched nanopores exploiting copper‐free click reactions and site‐specific protein conjugation systems. The functional immobilization of three different nanobodies is demonstrated in ligand binding experiments with green fluorescent protein, mCherry, and α‐amylase (α‐Amy) serving as molecular analytes. Ligand binding is resolved using a combination of optical and electrical recordings displaying quantitative dose–response curves. Furthermore, a change in surface charge density is identified as the predominant molecular factor that underlies quantitative dose–responses for the three different protein analytes in nanoconfined geometries. The devised strategy should pave the way for the systematic functionalization of nanopore surfaces with biological receptors and their ability to detect a variety of analytes for diagnostic purposes. Abstract : Synthetic nanopores carry great potential as physical transducers in biosensing. Yet, systematic strategies to functionalize their surface with proteins remain limited. Addressing these limitations, a general strategy for the orientated immobilization of proteins on the nanopore surface is presented. The approach exploits site‐specific protein conjugation tags to immobilize three different nanobodies to detect three distinct protein analytes in nanoconfined geometries.