Surface Plasmon Fluorescence Spectroscopy in Protein Binding Studies

W. Knoll

Surface plasmon fluorescence spectroscopy (SPFS) uses the greatly enhanced electromagnetic field of a surface-plasmon mode for the excitation of surface attached fluorophores, offering extraordinary sensitivity for monitoring interfacial binding interactions. Here, three model systems are presented sequentially to demonstrate the protein application of SPFS. The first example presents antibody-antigen interaction models built up on 2D and 3D surface architectures, mainly focusing on the application of SPFS in immunoassay. The second example concerns about building the supramolecular hierarchy based on bacterial S-layers. The third one describes coupling the light-harvesting complex II (LHCII) onto the surface and the potential of SPFS to investigate the energy transfer between neighboring LHCIIs.

SPFS is built on the basis of a Kretschmann surface plasmon spectroscopy (SPR or SPS) setup, adding a fluorescence detection unit which is aligned perpendicular to the gold or silver surface. An ultra-thin flow-cell is attached to the sensor surface, providing highly efficient sample delivery and greatly reduced sample consumption which favors biological application. Upon the binding of a fluorescent protein, both SPR and fluorescence signals can be offered by SPFS simultaneously, which is particularly interesting for protein studies. However in the kinetics mode, fluorescence signal could be ‘detuned’ by a significant SPR shift.

Fluorescence intensity profile of a fluorescent dye at the interface is a picture of surface plasmon evanescent field (as the excitation source) superimposed by the near-metal quenching profile. Thus, fluorescence yield is strongly dependent on the separation distance between dye and metal surface within ca. 20-30 nm. This, however, provides us an extra information to further understand steric arrangement of proteins at the interface. On the other hand, 3D interaction matrix can be employed to largely convolute the distance effect for practical sensing concerns. Its large loading capacity also favors highly sensitive detections.

2D matrices are built by mixed self-assembled monolayers (SAMs), providing tunable biotin (as an antigen) densities. Primary and secondary antibodies are bound to the surface sequentially, and their end-point SPR/fluorescence signals are plotted upon different biotin densities. Fluorescence signal doesn't scale linearly to the SPR signal, indicating that various molecular architectures were formed on different biotin surfaces.

3D matrices are commercially available CM5 chips from Biacore AB, Sweden. Large amount of mouse IgG (as an antigen) can be covalently attached to the 100 nm carboxymethyldextran (CMD) chains on the chip for the limit of detection (LOD) study of SPFS. Extremely diluted dye-labeled anti-mouse antibody solutions are incubated with the surface sequentially, followed by a regeneration step to clean up the fluorescent proteins. Operating with mass-transfer limited kinetics, the dose-response curve spans linearly over a large analyte concentration range (>6 orders of magnitude), and extends into the atto-molar (10-18) range, which corresponds to 5-10 molecules attaching to the sensing area in every minute.

Crystalline bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope proteins of prokaryotic organisms. Isolated S-layer subunits are capable of recrystallizing as closed monolayers onto solid supports. Furthermore, functional domains (e.g. core streptavidin) can be incorporated in S-layer proteins by genetic engineering, providing hundreds of strategies to build up versatile supramolecular hierarchies on the surface.

As the most abundant chlorophyll a/b binding protein, the light-harvesting complex associated with photosystem II (LHCII) accounts for half of the entire chlorophyll content in green plants. It functions to absorb light energy, transferring it to the reaction center, where the energy is then converted into stable chemical products. The biologically active complex also does the internal energy transfer from chl b to chl a, which is one type of Fluorescence Resonance Energy Transfer (FRET). To immobilize this easily denatured membrane protein complex on the surface while still maintaining its biological activity is the precondition to investigate the inter- and intra- energy transfer of LHCIIs.

A variety of techniques have been used to immobilize LHCIIs on silver or gold films. Among them, non-covalent specific binding is preferable in sensor application for its reversible characteristic. As a typical example in IMAC (Immobilized metal affinity chromatography), addition of six histidines to recombinant proteins has proved useful in their purification by nickel-NTA affinity columns. 6his-tagged apoprotein (LHCP) can be successfully overexpressed in Escherichia coli without influence on the function of the protein, which provides the possibility to study self fluorescent LHCIIs on the surface.