Controlled orientation and covalent attachment of proteins on biosensor surfaces by Chelation Assisted Photoimmobilization

• In the context of surface chemistry for affinity biosensor chips, it is widely accepted that uniform orientation of the immobilized recognition element (ligand) is preferred over random orientation. However, this assumption has often been based on studies where differences in ligand immobilization level have not been taken into account. In this contribution, we present a novel two-step method for homogenous orientation and covalent attachment of proteins to sensing surfaces, called Chelation Assisted Photoimmobilization (CAP). Careful quantification of the effect of ligand orientation on analyte responses was performed by comparing this strategy to immobilization by conventional amine coupling. In CAP, the chelation agent is nitrilotriacetic acid (NTA) which chelates Ni2+. A His-tagged ligand forms an oriented assembly when binding Ni2+-NTA and is then covalently bound to the surface via photolabile benzophenone (BP), which attacks C-H bonds upon UV light activation. We relied on a surface chemistry based on self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG)-containing alkanethiolates on gold. Alkanethiols terminated with either NTA, BP or OEG were synthesized and mixed SAMs were characterized by infrared reflection absorption spectroscopy (IRAS), ellipsometry and contact angle goniometry. IRAS was also used to quantify ligand immobilization levels obtained either by CAP or by amine coupling via the carboxyl groups of an NTA-presenting surface. The model ligand was human IgG-Fc modified with a C-terminal 6xHis-tag and the analyte was Protein A. The ligand-analyte interaction was quantified by a surface plasmon resonance biosensor. Analyte responses were normalized with respect to the ligand amounts obtained by the two immobilization strategies. Interestingly, the normalized analyte response with randomly oriented ligand was >2 times higher than that with ligand immobilized by CAP. This shows that oriented ligand immobilization is not necessarily a means of increasing the sensitivity of a biosensor. Factors that may influence performance include the valency of the ligand and constraints related to the surface chemistry used for orientation.

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