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Construction
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Construction

The base of the sensor is commonly a thin glass slide (BK7, N-SF6, D263T). On one side of the glass slide a metal layer is deposited which can generate the surface plasmons upon coupling with a p-polarized light beam. The metal layer consists generally of gold (~50 nm) but other metals such as Cu, Al and Ag are sometimes used. Between the glass and plasmonic metal layer, there is a very thin adhesion layer of Cr or Ti (~2 nm) because gold does not stick well to the glass layer. In addition, the adhesive layer also improves the stability of the gold layer (1), (2).

Since the metal (gold) layer is in most cases incompatible with biological solutions, the surface has to be protected against non-specific adsorption of proteins or other biological compounds. The pacifying layer should bind strongly to the gold layer and form a dense packing, effectively shielding the metal layer. In general, the pacifying layer – also referred to as a self-assembling monolayer (SAM) – has terminal functional groups which can be used for further modifications of the sensor surface.

Self-assembling monolayer

The attachment of a self-assembling monolayer on sensors with a gold layer is based on the strong adsorption of disulphides (R-S-S-R), sulphides (-S-R) and thiols (R-SH). The sulphur-containing dialkanes form highly ordered monolayers where the sulphur donor atoms coordinate strongly on the gold substrate and Van der Waals forces between methylene groups orient and stabilize the monolayer. The number of methylene groups regulates the ordering of the molecules, whereby chain lengths above 10 methylene groups assemble in a crystalline-like manner and shorter chain lengths are less ordered (3), (4), (5). The SAM can be used as a means to pacify the gold surface which will bind proteins very easily and can be used as a starting point for further modifications.

2D planar-like surfaces

Sensors with 2D planar-like surfaces are generally based on SAM's which are modified with small functionalizing groups (e.g. COOH) – as opposed to larger extensions, such as dextran, alginate or PEG – creating a more 3D structure. Therefore the 2D surface is less hydrophilic and may for some samples increase non-specific binding. The surface thickness is below 5 nm, offering a very flat surface (6). The flat surface may be of value in work with large analytes such as cells and virus particles, and can be used to test the influence of a matrix on kinetic determinations. The surface can be functionalized with, for instance, carboxyl, thiol or amine groups; however, the immobilization yield will be approximately 10% (~1–2 ng protein mm-2) of what can be obtained with a sensor with a carboxymethylated (dextran) matrix (50 ng mm-2) (7). In addition, on these surfaces the effect of pre-concentration is very low or is lacking all together. Higher ligand concentrations and longer immobilization times may be necessary to immobilize enough ligand.

3D-like surfaces

As opposed to the 2D-like structure the 3D-like surfaces are modified with compounds that give the surface a volume (thickness) and more possibilities (capacity) to attach molecules. One of the most common 3D matrices used is carboxymethylated dextran.
Starting with a gold-coated glass slide, the sensor surface is treated with a solution of 16-mercapto-hexadecan-1-ol (5 mM) in ethanol water (80:20). The resulting hydrophilic surface is reacted with 0.6 M epichlorohydrin in a 1:1 mixture of 0.4 M NaOH and diglycine for 4 hours at 25°C. This step will introduce epoxy groups on the surface, which will serve as electrophilic coupling sites. After thorough washing with water, ethanol and water, the surface is treated with a dextran solution of 30% dextran (Mr 500,000 Da) in 0.1 M NaOH for 20 hours at 25°C. After washing, the surface is reacted with 1 M bromoaceticacid in 2 M NaOH for 16 hours at 25°C. This will result in approximately one carboxyl group per glucose unit. After thorough washing, the surface is ready for further use (8), (9). The carboxyl groups are the starting point for further functionalisations.

References

(1)Ekgasit, S., C. Thammacharoen, F. Yu, et al. Influence of the Metal Film Thickness on the Sensitivity of Surface Plasmon Resonance Biosensors. Applied Spectroscopy 59: 661-667; (2005). Goto reference
(2)Sexton, B. A., B. N. Feltis and T. J. Davis Characterisation of gold surface plasmon resonance sensor substrates. Sensors and Actuators A: Physical 141: 471-475; (2008). Goto reference
(3)Nuzzo, R. G. and D. L. Allara Adsorption of bifunctional organic disulfides on gold surfaces. Journal of the American Chemical Society 105: 4481-4483; (1983). Goto reference
(4)Porter, M. D., T. B. Bright, D. L. Allara, et al. Spontaneously organized molecular assemblies. 4. Structural characterization of n-alkyl thiol monolayers on gold by optical ellipsometry, infrared spectroscopy, and electrochemistry. Journal of the American Chemical Society 109: 3559-3568; (1987). Goto reference
(5)Wink, T., S. J. van Zuilen, A. Bult, et al. Self-assembled monolayers for biosensors. Analyst 122: 43R-50R; (1997).
(6)XanTec XanTec bioanalytics GmbH. (2012). Goto reference
(7)Johnsson, B., S. Lofas and G. Lindquist Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Analytical Biochemistry 198: 268-277; (1991).
(8)Lofas, S. and B. Johnsson A novel hydrogel matrix on gold surfaces in surface plasmon resonance sonsors for fast en efficient covalent immobilization of ligands. J.chem.soc., chem commun. 1526-1528; (1990).
(9)Lofas, S. Dextran modified self-assembled monolayer surfaces for use in biointeraction analysis with surface plasmon resonance. Pure & Appl.Chem. 67: 829-834; (1995).