TY - JOUR
T1 - Reversible Immobilization of Proteins in Sensors and Solid-State Nanopores
AU - Ananth, Adithya
AU - Genua, María
AU - Aissaoui, Nesrine
AU - Díaz, Leire
AU - Eisele, Nico B.
AU - Frey, Steffen
AU - Dekker, Cees
AU - Richter, Ralf P.
AU - Görlich, Dirk
N1 - Accepted Author Manuscript
PY - 2018/5/3
Y1 - 2018/5/3
N2 - 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.
AB - 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.
KW - biosensing
KW - histidine tag
KW - nanopores
KW - selective immobilization
KW - sensors
KW - surface functionalization
UR - http://www.scopus.com/inward/record.url?scp=85044715892&partnerID=8YFLogxK
U2 - 10.1002/smll.201703357
DO - 10.1002/smll.201703357
M3 - Article
AN - SCOPUS:85044715892
SN - 1613-6810
VL - 14
JO - Small
JF - Small
IS - 18
M1 - 1703357
ER -