Download technical note 123 spr imaging sensitivity

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
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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.
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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.
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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.
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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:
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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.
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This corresponds to 16 femtomoles mm-2;
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The molecular mass of the 18-mer oligo analyte is ~5940;
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16 femotomoles of 18-mer oligo thus corresponds to 95 picograms mm-2;
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That is, a 0.6% reflectivity change corresponds to a mass change of 95 pg mm ;
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More simply, a 1% reflectivity change indicates a mass change of ~158 pg mm-2;
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The limit of detection for the SPRimager®II is a response change of ~0.05% refelctivity;
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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