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TECHNICAL NOTE 123 SPR IMAGING SENSITIVITY Summary Sensitivity of Surface Plasmon Resonance (SPR) imaging depends ultimately on the ability of a detection system to discriminate changes in mass density on a surface. In practice, sensitivity depends significantly on the nature of the molecular interaction under study, and the probe immobilization strategy used. GWC’s open system allows complete freedom in choice of chemistry. This, together with the tremendous versatility of label-free SPR detection, facilitates analysis of a very broad range of interactions. Factors influencing sensitivity of SPR measurements • The affinity of the molecular interaction. The higher the affinity, the smaller the concentration of analyte that can be detected. Thus it is easier, for example, to detect a high affinity interaction such as a biotinylated analyte binding to a streptavidin probe, than it is to detect a lower affinity interaction such as a pheromone binding to its receptor. • The mass of the analyte. The more mass that binds to the surface probes, the greater the SPR response. This means that higher MW molecules such as proteins are more readily detectable on a molar concentration basis than smaller molecules such as hormones. Likewise, bigger proteins are more readily detected than smaller proteins for a given interaction affinity. • The density of the probe. The more functional probe molecules that are immobilized per unit of surface area, the more mass of analyte can be adsorbed, and the greater the SPR response. Thus the biosensor surface is critical for sensitivity. More densely packed probes normally deliver greater sensitivity, but for larger analytes, or for molecules with multiple binding sites, steric effects may mean that improved sensitivity is obtained with lower probe molecule densities. Using GWC’s open systems, users may select surface chemistry approaches that allow control over probe density on the chip surface. This contrasts with approaches that use entangled polymers such as dextran to immobilize probes, where probe densities are difficult or impossible to predict. • The robustness of controls. Controls for non-specific binding of analyte are usually essential, and experimental changes must be corrected for any change in control response. Probe arrays offer the most robust control mechanism, as one or more probes on the same surface as the experimental probes can be used as positive or negative controls. Comparing SPR systems Comparing the performance of different SPR systems is extremely difficult. Due to different designs, it is virtually impossible to create identical biosensors for two distinct instruments, and since the biosensor surface is critical in determining sensitivity, comparing sensitivies objectively is thus challenging. Ideally, fundamental physical units would be used to compare SPR system sensitivities. GWC’s systems allow direct measurement of reflectivity changes, unlike most other systems, which use non-fundamental units that are valid only on a given instrument. Most manufacturers of SPR systems do not publish reflectivity values, as this fundamental measurement cannot be obtained with most instruments. A popular alternative is to quote sensitivity in terms of the minimum refractive index difference in the "bulk solution" that the instrument can distinguish. (The bulk solution is the liquid flowed over the surface of the SPR chip.) However, the change in refractive index of the bulk solution bears little relation to the measurement sought. In fact, the SPR response of interest results from changes in refractive index change that occur at the surface, where the probe interacts with the analyte, and not in the solution above. GWC therefore does not use refractive index changes of bulk solutions as a measure of sensitivity. Minimum Change in Mass Density Detectable Estimates of the minimum change in mass density that can be detected using the SPRimager®II can be made from the published literature. For example, Nelson et al. (Anal. Chem. 2001, 73, 1-7) measured SPRimager® performance in nucleic acid hybridization experiments. Thiolated DNA probes were immobilized on gold surfaces via Self-Assembled Monolayers (SAMs), which provide for predictable probe densities on the sensor surface. We can make the following extrapolations from Nelson et al. to obtain an estimate of sensitivity: • Nelson et al. estimated that a 0.6% change in reflectivity is obtained on the SPRimager® when 4 femtomoles of 18-mer oligonucleotide is adsorbed in a probe spot 0.5mm square. > This corresponds to 16 femtomoles mm-2; > The molecular mass of the 18-mer oligo analyte is ~5940; > 16 femotomoles of 18-mer oligo thus corresponds to 95 picograms mm-2; > -2 That is, a 0.6% reflectivity change corresponds to a mass change of 95 pg mm ; > More simply, a 1% reflectivity change indicates a mass change of ~158 pg mm-2; > The limit of detection for the SPRimager®II is a response change of ~0.05% refelctivity; > Therefore the limit of detection is a change of [158*0.05 pg / sq. mm] = ~8 pg mm-2. These numbers are built on an assumption that the probe density in the experiments of Nelson et al. was optimal at 1012 molecules mm-2, and that all probe molecules capture homologous oligo analyte. However, if either assumption is incorrect, the sensitivity of the instrument would be better than the above estimate. Dynamic Range The SPR response is measured as a change in reflectivity of areas on the surface of the array on the SPRimager®II. Changes in reflectivity are linear with respect to mass density changes on the surface up to a 10% change in reflectivity (Nelson et al., 2001—see plot at right), which is ample for most molecular interaction experiments. Thus the linear dynamic range is measurement of changes in reflectivity of 0.05% to 10%. Larger reflectivity changes may still be monitored with the SPRimager®II, but linearity with respect to mass density change will fall off. Since increases in mass on the surface lead to increased SPR reflectivity, regions of the array with responses exceeding the instrument’s dynamic range will always be obvious as regions of very high reflectivity. If such responses are problematic, probe density can be adjusted downwards to bring the system back into dynamic range. Theoretical change in percent reflectivity as a function of changes in refractive index generated by a DNA probe region immobilized on gold. For more information and for protocols for specific applications, please contact your GWC Technologies representative. www.gwctechnologies.com [email protected] 608.441.2721