Detecting Selective Protein Binding Inside Plasmonic Nanopores: Toward a Mimic of the Nuclear Pore Complex

Abstract

Biosensors based on plasmonic nanostructures offer label-free and real-time monitoring of biomolecular interactions. However, so do many other surface sensitive techniques with equal or better resolution in terms of surface coverage. Yet, plasmonic nanostructures offer unique possibilities to study effects associated with nanoscale geometry. In this work we use plasmonic nanopores with double gold films and detect binding of proteins inside them. By thiol and trietoxysilane chemistry, receptors are selectively positioned on the silicon nitride interior walls. Larger (~150 nm) nanopores are used detect binding of averaged sized proteins (~60 kg/mol) with high signal to noise (>100). Further, we fabricate pores that approach the size of the nuclear pore complex (diameter down to 50 nm) and graft disordered phenylalanine-glycine nucleoporin domains to the walls, followed by titration of karyopherinβ1 transport receptors. The interactions are shown to occur with similar affinity as determined by conventional surface plasmon resonance on planar surfaces. Our work illustrates another unique application of plasmonic nanostructures, namely the possibility to mimic the geometry of a biological nanomachine with integrated optical sensing capabilities.

Surface coatings for solid-state nanopores

“…288,292 In the context of nanopore sensing, SAMs are predominantly used for sensing specific analytes, 193,201,203,204 minimizing non-specific interactions, 188,197,200,202,208 manipulating surface charge, 192,194,[196][197][198] and adding functionality such as gating of the pore, 192,205,206 preferential transport, 194,[196][197][198][199] or enhancing the signal of plasmonic nanopores. 207,209 Charles R. Martin's group was among the first to take advantage of SAMs in nanopores by chemisorbing thiols to gold surfaces deposited onto track etched nanotubes. 195 The same group has since explored other modifications involving SAMs.…”

Transport receptor occupancy in nuclear pore complex mimics

“…A major difficulty in settling the debate stems from the limits in spatiotemporal resolution of imaging techniques [1,3,41], combined with the complexity of the NPC in its physiological state, as it features a central mesh of ~ 200 unstructured FG-Nups that are confined into a ~ 45-70 nm pore that is constantly being crossed by many types of NTR-cargo complexes in large numbers (~ 10 3 of such protein complexes per second per pore [41]) in both directions. To probe nuclear transport through the FG-mesh, artificial mimics of the NPC have been successfully created that recapitulate the selective binding and transport behavior observed in vivo [23,[42][43][44][45][46][47][48][49][50][51][52]. Prominent examples are biomimetic nanopores, in which ~ 30-50 nm solid-state nanopores are chemically functionalized using a single type of FG-Nup (e.g., Nsp1 or Nup98) and translocations of Kaps through the reconstituted FG-mesh are monitored optically [42] or electrically [45,53,54].…”

Selectively detecting attomolar concentrations of proteins using gold lined nanopores in a nanopore blockade sensor

“…16 The chemical functionalities on the functionalized nanopore chips can be removed via oxygen plasma treatment and regenerated for multiple times. There are only a few attempts [28][29][30][31] at obtaining site-selective modication on solid-state nanopores that typically use colloidal lithography to fabricate the nanopores and either used sacricial layers to place surface chemistry on one location and not the other 28 or had a layered structure of two materials such that there were well dened layers of surface chemistry within the nanopore. 29,30 The fabrication approach reported herein is a further advance over these capabilities because rstly it can produce ordered arrays of nanopores where the fabrication approach presents the control over nanopore numbers, and diameters and positions that are modulable for individual metallic nanopores or even with a mixture of metallic and semiconducting nanopores, the entire inner pore walls can be modied with the same surface chemistry which is different to the exterior surface and as there is no further fabrication processing even biological and delicate chemistries can be used.…”