Reversible Immobilization of Proteins in Sensors and Solid‐State Nanopores

Abstract

The controlled functionalization of surfaces with proteins is crucial for many analytical methods in life science research and biomedical applications. Here, a coating for silica-based surfaces is established which enables stable and selective immobilization of proteins with controlled orientation and tunable surface density. The coating is reusable, retains functionality upon long-term storage in air, and is applicable to surfaces of complex geometry. The protein anchoring method is validated on planar surfaces, and then a method is developed to measure the anchoring process in real time using silicon nitride solid-state nanopores. For surface attachment, polyhistidine tags that are site specifically introduced into recombinant proteins are exploited, and the yeast nucleoporin Nsp1 is used as model protein. Contrary to the commonly used covalent thiol chemistry, the anchoring of proteins via polyhistidine tag is reversible, permitting to take proteins off and replace them by other ones. Such switching in real time in experiments on individual nanopores is monitored using ion conductivity. Finally, it is demonstrated that silica and gold surfaces can be orthogonally functionalized to accommodate polyhistidine-tagged proteins on silica but prevent protein binding to gold, which extends the applicability of this surface functionalization method to even more complex sensor devices.

Surface coatings for solid-state nanopores

“…286 In the absence of polymerization, silanization forms thin coatings with low surface density, which may be used to increase hydrophobicity 185,190 or to reduce non-specific surface adhesion. 283 In the context of nanopores, silanization allows for the functionalization of pore walls by enabling the attachment of DNA, 173,175,178,183 dendrimers, 174 nucleoporins, 176 aldehydes, 172,177 spiropyran moieties, 181 cysteines, 187 carboxylic acid, 172 EDTA, 188 peptides, 189,191 and polymer brushes 182 to chemical groups that are attached to the silane molecule. Apart from the possibility of such attachments, silanization can generate a coating with antifouling properties 184 and can be used to manipulate ICR 185 and other charge-based properties, 179 including the modulation of surface charge by changing the pH value of the recording electrolyte 129,180,186 Fig .…”

“…Apart from gold deposition, SAM preparation does not require specialized equipment, the monolayers can form over large surfaces and they can provide surface groups that repel molecules, interact with, or covalently link to molecules of interest. 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.…”

Microparticle Delivery of Protein Markers for Single‐Cell Western Blotting from Microwells

“…To meet the particle‐loading metric, we choose magnetic particles so that we might use an applied, external magnetic field for directed loading of the microparticles into cell‐laden microwells . To meet the more stringent triggered release metric, we utilize microparticles coated with Ni 2+ for reversible binding of histidine(His)‐tagged proteins, which can then be chemically released from the microparticle concurrently with a 30 s cell lysis step …”

Nanopore‐Based, Rapid Characterization of Individual Amyloid Particles in Solution: Concepts, Challenges, and Prospects

“…In general, synthetic nanopores enable direct monitoring of changes in the populations of oligomeric species and of large natively folded monomers ( Figure ). Recent years indicate a shift toward synthetic nanopores for amyloid sensing ( Table 5 ), and we expect this trend to continue with the development of low‐noise recording setups, high‐bandwidth recording equipment, and advanced surface coating technologies that may selectively bind amyloids. Because of their single‐molecule sensitivity and broad size range, synthetic nanopores may ultimately provide insight into the ways in which these amyloid‐forming proteins aggregate, their size distributions, and their structures.…”