Bare gold sensors are not of much use in SPR. Therefore the gold surface is covered with a self-assembling monolayer (SAM) of an alkanethiol that is the starting point of further sensor modifications. The functionalization needed can be attached directly to the SAM or there can be a matrix that functions as an intermediate layer to reduce non-specific binding. The added benefit of a matrix is that there are more sites where the new binding groups can be attached.
The most common functionalization is the attachment of a dextran matrix. Depending on the length of the dextran chains used for coating the thickness of the matrix layer is between 30–500 nm. Other matrices can range from agarose, alginate or gelatine gel, polyethylene glycol (PEG), poly-L-lysine, carboxymethyl cellulose or a linear polycarboxylate hydrogel (1),(2). Alginate is a linear copolymer with homopolymeric blocks of (1–4)-linked β-D-mannuronate and its C-5 epimer α-L-guluronate residues, respectively, covalently linked together in different sequences or blocks.
A drawback of a 3D matrix is that both the ligand during the immobilization and the analyte during the measurements have to penetrate into the matrix. A 3D matrix can enhance mass transfer limitation and thereby mask fast kinetics.
Carboxylation is the most common modification on a sensor chip. This versatile moiety can be modified in various ways to suit the immobilization needs such as amine, thiol and aldehyde coupling. Each of these procedures starts with the activation of the carboxyl group with a mixture of NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N’-(dimethylaminopropyl)carbodiimide) to create N-Hydroxysuccinimide esters. By varying the activation time, more or fewer carboxyl groups are activated. In addition, the concentration of the NHS/EDC mixture can be varied to control the quantity of activated carboxyl groups. The N-Hydroxysuccinimide esters can be directly reacted with (primary)amine groups on for instance proteins to form a covalent bond between the sensor surface and protein. In the case of the ligand thiol method, the activated surface is first incubated with a reactive disulphide group and then with a thiol on a ligand. Likewise, with the aldehyde coupling the activated group is first modified with a hydrazide group and then incubated with an ligand with free aldehyde groups.
An additional effect of a 3D carboxylated matrix is that the pre-concentration effect can be exploited to generate efficient immobilization conditions and thereby minimize sample consumption.
A sensor with pre-immobilized (strept)avidin or NeutrAvidin™ is a general-purpose sensor for capture of biotinylated ligands. A combination of high affinity between avidin and D-biotin (KD ~10-15) or streptavidin and D-biotin (KD 10-13) (3), binding capacity, reproducibility and chemical resistance provides excellent performance in a broad range of applications. This kind of sensor surface achieves a high binding capacity over a broad molecular mass range.
Sensor surfaces can be pre-immobilized with biotin that subsequently can bind to (strept)avidin, effectively creating a (strept)avidin surface that can bind biotinylated ligands (biotin-avidin-biotin bridging).
Protein A,G, L
In many applications the bacterial derived proteins A, G and L are used to bind antibodies (4). When, for instance, protein A is immobilized on a sensor it can function as a ligand capture molecule and can capture antibodies from crude solutions such as culture media. In the meantime, the antibodies will be properly orientated since the capture is predominately via the Fc part of the antibodies (5).
Because the antibody capture is a transient interaction, the sensor surface can be regenerated and subsequently other antibodies can be captured which greatly enhances the lifetime of the sensor. A drawback of the antibody capture by protein A, G or L can be the dissociation of the antibodies during the experiments which causes the surface to decay during measurements(6),(7).
Sensor surfaces modified with nitrilotriacetic acid (NTA) are designed to bind histidine-tagged molecules (His6-tag). The binding of the histidine chains relies on an NTA-chelated metal ion. The most commonly used are Cu2+, Ni2+, Zn2+ and Co2+ ions which exhibit varying affinities and specificities towards histidines (8). In general, the affinity (nickel: KD ~ 10-6 M) (9),(10) of this interaction is sufficiently high to allow for detailed analysis of subsequent analyte binding. Immobilization via His-tags also has the advantage of orientating the ligand molecules in a homogeneous way and allows immobilization without significantly changing the pH or ionic strength during the coupling procedure. The captured ligand is relatively easy to remove using Imidazole – a competitive interaction with the NTA – or by stripping out the metal ion with EDTA. After subsequently loading new metal ions, the surface can be reused.
Hydrophobic surfaces are designed for studies with lipid monolayers. The surfaces can be used to study how proteins interact with the lipid layer or to study how analytes interact with membrane-bound receptors. In addition, hydrophobic surfaces can be used to investigate direct hydrophobic adsorption of the ligand, for instance, to mimic an ELISA.
Lipophilic surfaces are used to capture vesicles and to mimic lipid bilayers. The lipid bilayer makes it possible to investigate membrane-incorporated proteins and trans-membrane proteins.
|(1)||XanTec XanTec bioanalytics GmbH. (2020). Goto reference|
|(2)||Bio-Rad ProteOn sensor chips - overview - bulletin 5404D. (2012).|
|(3)||Vermette, P., T. Gengenbach, U. Divisekera, et al. Immobilization and surface characterization of NeutrAvidin biotin-binding protein on different hydrogel interlayers. Journal of Colloid and Interface Science 259: 13-26; (2003). Goto reference|
|(4)||Makaraviciute, A. and A. Ramanaviciene Site-directed antibody immobilization techniques for immunosensors. Biosensors and Bioelectronics 50: 460-471; (2013). Goto reference|
|(5)||Catimel, B., M. Nerrie, F. T. Lee, et al. Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor. A comparison of immobilisation strategies. J.Chromatogr.A. 776: 15-30; (1997).|
|(6)||Joss, L., T. A. Morton, M. L. Doyle, et al. Interpreting kinetic rate constants from optical biosensor data recorded on a decaying surface. Analytical Biochemistry 261: 203-210; (1998). Goto reference|
|(7)||Palau, W. and C. Di Primo Single-cycle kinetic analysis of ternary DNA complexes by surface plasmon resonance on a decaying surface. Biochimie 94: 1891-1899; (2012). Goto reference|
|(8)||Gaberc-Porekar, V. and V. Menart Perspectives of immobilized-metal affinity chromatography. Journal of Biochemical and Biophysical Methods 49: 335-360; (2001). Goto reference|
|(9)||Nieba, L., A. S. E. Nieba, A. Persson, et al. BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Analytical Biochemistry 252: 217-228; (1997).|
|(10)||Knecht, S., D. Ricklin, A. N. Eberle, et al. Oligohis-tags: mechanisms of binding to Ni2+-NTA surfaces. (2009). Goto reference|