Biomaterials Tutorial

Surface Plasmon Resonance (SPR)

Janet Cuy
University of Washington Engineered Biomaterials

Surface plasmon resonance (SPR) is a non-destructive analysis technique, useful for investigating thin layers of molecules upon a material surface.  Specifically, SPR is capable of detecting changes in refractive index (n) occurring near the surface of a metal (within ~200nm) [1].  Refractive index refers to the speed with which light passes through a material compared to the speed with which it passes through air.  For example, light travels more slowly through glass than it does through air, therefore glass has a higher refractive index than air (nglass=1.5-1.6, nair=1) [2].

The use of a metal sensing surface in SPR is critical, as this technique capitalizes upon the fact that metals (like other electrically conductive materials) contain electrons that behave as a continuous "sea" of charge.  This "sea" of charge can undergo charge-density oscillations (or plasmons) at the surface of the conductor, particularly at a surface in contact with an insulator [3, 4, 5].  Following the "sea" analogy, imagine the behavior of seaweed washing up against a shore: the density of seaweed (i.e., electrons) at the shore (the interface between the "conducting" water and the "insulating" sand) varies with the ebb and flow of the tide (the plasmon).

A simple SPR instrument set-up generally consists of a light source, a glass prism with a high refractive index n, a thin (50 nm) metal film placed in contact with the bottom of the prism and a photodetector (see Figure 1) [6].  The molecular layer of interest can be coated onto the thin metal film may be coated with a molecular layer of interest on the side opposite the prism.

Figure 1.  Basic SPR instrument configuration.  Adapted from Levesque and Paton [6].

Surface plasmon waves (SPWs) can be generated at the interface between the conductive metal film and the insulating molecular layer by striking the metal sensor with a particular type of light [7, 8].  At the same time that SPWs are generated, light is also reflected off of the metal surface.  Past a specific incident angle (q in Figure 1), and only in the presence of the highly refractive glass prism, all the energy from the incident light wave will be transferred to the reflected light wave (total internal reflection) [9].  However, at  a very specific angle past the point of total internal reflection (the SPR angle), a majority of the incident light energy that would have typically been transferred to the reflected light wave will instead interact with the generated SPWs, resulting in a phenomenon called resonance [6, 10].  At resonance, a minimum in reflected light intensity will be observed, and the SPR angle can thus be determined by measuring the intensity of the reflected light (via photodetector), and plotting it as a function of incidence angle (see Figure 2) [10].

Figure 2.  Example of SPR spectrum.  Adapted from Kolomenskii, et al. [11].

The SPR angle is dependent on several factors, including: characteristics of the metal film, the incident light, and the thickness and refractive index of the molecular layer in contact with the metal sensing surface [10].  Consequently, spectra can be generated for a metal surface with and without a coated molecular layer. Then, the shift in SPR angle between the two can be quantified and used to calculate the thickness or refractive index of the adhered molecules [12].  SPR has proven useful in determining both growth in the thickness of a molecular layer [13] and loss in thickness, even of a single monolayer [14].

Along with its ability to determine the thickness of coated films, SPR has also emerged as a technology in the area of sensors (e.g., for the detection of physical quantities, chemicals and biologics) [8].  Physical quantities (such as temperature and humidity) can be deduced from changes in refractive index.  Chemical sensing can use changes in refractive index to indicate changing concentrations of molecules adhered to the metal surface (as a result of chemical reactions).  Biosensing can also use refractive index changes to deduce the occurrence of binding interactions (such as between antigens and antibodies).  SPR also provides the important advantage of being able to monitor reactions in real-time, without the need to go through the often complicated process of labeling molecules with fluorescent or radioactive probes [15].

Like all surface analysis techniques, SPR has its limitations in terms of sensitivity (the smallest amount of molecule detectable) [5, 8], resolution (the smallest difference in SPR angle distinguishable) [8, 16] and sample characteristics (geometry, thickness, etc.).  However, this technique still provides a remarkable variety of capabilities for the characterization of reaction kinetics and thin film properties, with a high degree of sensitivity and in real-time—all important factors for a biomaterials scientist involved in the engineering, alteration and study of functionalized surfaces.

References:

1. Sigal GB, Mrksich M, Whitesides GM. Using surface plasmon resonance spectroscopy to measure the association of detergents with self-assembled monolayers of hexadecanethiolate on gold. Langmuir 1997; 13: 2749-2755.
2. Foster B. Optimizing light microscopy for biological and clinical laboratories. Dubuque: Kendall/Hunt; 1997. p. 5.
3. Raether H. Surface plasma oscillations and their applications. Phys Thin Films 1977; 9: 145-244.
4. Ratner BD, Castner DG. Electron spectroscopy for chemical analysis. In: Vickerman JC, editor. Surface analysis: The principal techniques. New York: John Wiley&Sons, 1997.
5. Garland PB. Optical evanescent wave methods for the study of biomolecular interactions. Q Rev Biophys 1996; 29: 91-117.
6. Levesque L, Paton BE. Detection of defects in multiple-layer structures by using surface plasmon resonance. Appl Opt 1997; 36: 7199-7203.
7. Caruso F, Jory MJ, Bradberry GW, Sambles JR, Furlong DN. Acousto-optic surface-plasmon resonance measurements of thin films on gold. J Appl Phys 1998; 83: 1023-1028.
8. Homola J, Yee SS, Gauglitz G. Surface plasmon resonance sensors: Review. Sensors Actuators B:Chem 1999; 54: 3-15.
9. Fishbane PM, Gasiorowicz S, Thornton ST. Physics for scientists and engineers. Englewood Cliffs: Prentice Hall, 1993.
10. Stenberg E, Persson B, Roos H, Urbaniczky C. Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J Colloid Interface Sci 1991; 143: 513-526.
11. Kolomenskii AA, Gershon PD, Schuessler HA. Sensitivity and detection limit of concentration and adsorption measurements by laser-induced surface-plasmon resonance. Appl Opt 1997; 36: 6539-6547.
12. de Bruijn HE, Kooyman RPH, Greve J. Determination of dielectric permittivity and thickness of a metal layer from a surface plasmon resonance experiment. Appl Opt 1990; 29: 1974-1978.
13. Caruso F, Niikura K, Furlong DN, Okahata Y. Ultrathin multilayer polyelectrolyte films on gold: construction and thickness determination. Langmuir 1997; 13: 3422-3426.
14. Herminghaus S, Leiderer P. Nanosecond time-resolved study of pulsed laser ablation in the monolayer regime. Appl Phys Lett 1991; 58: 352-354.
15. Geddes NJ, Martin AS, Caruso F, Urquhart RS, Furlong DN, Sambles JR et al.Immobilisation of IgG onto gold surfaces and its interaction with anti-IgG studied by surface plasmon resonance. J Immunol Methods 1994; 175: 149-160.
16. Tao NJ, Boussaad S, Huang WL, Arechabaleta RA, D'Agnese J. High resolution surface plasmon resonance spectroscopy. Rev Sci Instrum 1999; 70: 4656-4660.