Introduction to Surface Plasmon Resonance

ANNA J. TUDOS AND RICHARD B.M. SCHASFOORT

1.1 What is Surface Plasmon Resonance?

Since its first observation by Wood in 1902, the physical phenomenon of surface plasmon resonance (SPR) has found its way into practical applications in sensitive detectors, capable of detecting sub-monomolecular coverage. What is surface plasmon resonance? Wood observed a pattern of "anomalous" dark and light bands in the reflected light, when he shone polarized light on a mirror with a diffraction grating on its surface. Physical interpretation of the phenomenon was initiated by Lord Rayleigh, and further refined by Fano, but a complete explanation of the phenomenon was not possible until 1968, when Otto and in the same year Kretschmann and Raether reported the excitation of surface plasmons. Application of SPR-based sensors to biomolecular interaction monitoring was first demonstrated in 1983 by Liedberg et al. A historical overview of the use of the phenomenon for biosensor applications is given in Section 1.3 of this chapter. To understand the excitation of surface plasmons, let us start with a simple experiment.

1.1.1 A Simple Experiment

Consider the experimental set-up depicted in Figure 1.1. When polarized light is shone through a prism on a sensor chip with a thin metal film on top, the light will be reflected by the metal film acting as a mirror. On changing the angle of incidence, and monitoring the intensity of the reflected light, the intensity of the reflected light passes through a minimum (Figure 1.1, line A). At this angle of incidence, the light will excite surface plasmons, inducing surface plasmon resonance, causing a dip in the intensity of the reflected light. Photons of p-polarized light can interact with the free electrons of the metal layer, inducing a wave-like oscillation of the free electrons and thereby reducing the reflected light intensity.

The angle at which the maximum loss of the reflected light intensity occurs is called resonance angle or SPR angle. The SPR angle is dependent on the optical characteristics of the system, e.g. on the refractive indices of the media at both sides of the metal, usually gold. While the refractive index at the prism side is not changing, the refractive index in the immediate vicinity of the metal surface will change when accumulated mass (e.g. proteins) adsorb on it. Hence the surface plasmon resonance conditions are changing and the shift of the SPR angle is suited to provide information on the kinetics of e.g. protein adsorption on the surface.

1.1.2 From Dip to Real-time Measurement

Surface plasmon resonance is an excellent method to monitor changes of the refractive index in the near vicinity of the metal surface. When the refractive index changes, the angle at which the intensity minimum is observed will shift as indicated in Figure 1.2, where (A) depicts the original plot of reflected light intensity vs. incident angle and (B) indicates the plot after the change in refractive index. Surface plasmon resonance is not only suited to measure the difference between these two states, but can also monitor the change in time, if one follows in time the shift of the resonance angle at which the dip is observed. Figure 1.2 depicts the shift of the dip in time, a so-called sensorgram. If this change is due to a biomolecular interaction, the kinetics of the interaction can be studied in real time.

SPR sensors investigate only a very limited vicinity or fixed volume at the metal surface. The penetration depth of the electromagnetic field (so-called evanescent field) at which a signal is observed typically does not exceed a few hundred nanometers, decaying exponentially with the distance from the metal layer at the sensor surface. The penetration depth of the evanescent field is a function of the wavelength of the incident light, as explained in Chapter 2.

SPR sensors lack intrinsic selectivity: all refractive index changes in the evanescent field will be reflected in a change of the signal. These changes can be due to refractive index difference of the medium, e.g. a change in the buffer composition or concentration; also, adsorption of material on the sensor surface can cause refractive index changes. The amount of adsorbed species can be determined after injection of the original baseline buffer, as shown in Figure 1.2. To permit selective detection at an SPR sensor, its surface needs to be modified with ligands suited for selective capturing of the target compounds but which are not prone to adsorbing any other components present in the sample or buffer media.

1.2 How to Construct an SPR Assay?

Now we have a basic understanding of the surface plasmon resonance signal and how to measure it in time. We know that the sensor surface needs to be modified to allow selective capturing and thus selective measurement of a target compound. In the following, we are going to learn more about an SPR measurement. First, the steps of an SPR assay will be discussed from immobilization through analysis to regeneration in a measurement cycle. Next, we get acquainted with a typical calibration curve, followed by examples of assay formats. Finally, a short outlook is provided on the basics of the instrumentation.

1.2.1 The Steps of an Assay

In the simplest case of an SPR measurement, a target component or analyte is captured by the capturing element or so-called ligand (Figure 1.3). The ligand is permanently immobilized on the sensor surface previous to the measurement. Various sensor surfaces with immobilized ligands are commercially available, and many more can be custom-made, as explained in Chapters 6 and 7.

In the simplest case, the event of capturing the analyte by the ligand gives rise to a measurable signal, this is called direct...