Sensitivity

SPR instrument sensitivity

Many SPR instruments measure only relative changes, whether it is angle, wavelength or intensity. Overall, the optimal way to compare instrument performance is to run them on the same sample side-by-side, as documented in BioNavis White Paper (comparison between BioNavis 420A ILVES and Biacore T200).



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to traditional SPR in Protein Research



There are certain technological limitations which make different configurations more or less sensitive by default. For instance, fixed angle and camera-based detection make imaging SPR much less sensitive as compared to traditional SPR or MP-SPR. 

The sensitivity of SPR instruments is given by the wavelength at which they measure - lower wavelength equates to larger shift, resulting in greater sensitivity and smaller sample penetration depth. Sensitivity is also denoted by sensor layer materials and optionally their nanopatterning, as sharper SPR peaks lead to a reduction in the noise caused by peak min determination.

In summary, all SPR instruments measure only changes in refractive index (RIU). A simple test based on RI change (e.g. injection of small concentration of ethanol or glycine) can determine the sensitivity of the instrument. However, one can only truly compare instruments based on a specific assay.

 

 

 

Unit conversions (useful when comparing different instruments)

RIU Refractive Index Unit, a physical value representing the change in refractive index
RU The unit established for Biacore™ instruments. A rule of thumb is 1 RU~0.1 mdeg~1 pg/mm2
mdeg The SPR peak position shift upon change of refractive index
pg/mm2 Surface coverage can be calculated if one knows the parameters of the studied protein. This requires several assumptions and so is not straightforward. The response in RU or mdeg is proportional to surface coverage

 

Table on SPR unit conversions

Biacore         BioNavis   Generic Surface coverage
(approx. for typical protein)
790 nm 785 nm 670 nm
RU mdeg mdeg μRIU pg/mm2
1 0.1 0.12 1 1

 

Analytical sensitivity - limit of detection (LOD)

The limit of detection (LOD) is mostly instrument and assay specific thus might be referred as analytical sensitivity. For the instrument sensitivity, the most defining property is the signal-to-noise ratio, in practice the smallest detectable change in refractive index (for optical techniques). The LOD is typically three times standard deviation of the signal noise over sensitivity.

The overall performance of the instrument, and therefore the minimal detectable concentration of e.g. a protein, is influenced by several factors. These include the instrument setup (optics, fluidics and temperature control) and the protocol, such as flow rate and contact time between the binder and the sample. In the best case, SPR can detect less than a monolayer of large molecules (> 1000 Da). On a special surface, increasing surface-to-volume ratio (such as carboxymethyl dextrans), small molecules (< 1000 Da) can be measured directly. However, in concentration assays, the indirect or inhibition assay format is often used. Crucially, the performance is impacted by the assay at hand, impacted by affinity of the binding pair, non-specific binding from a complex matrix, and quality and purity of binders.


A: Direct assay. Left: Sensor is prepared with a layer of ligands. Middle: When sample is introduced, analyte binds directly to the ligand and generates a signal that is directly proportional to the concentration (right)


B: Indirect assay (also known as inhibition or competitive assay). Left: Sensor is coated with an analyte. Middle: Sample including free analyte is mixed together with antibody and introduced into the system. The more there is of free analyte (original sample), the less there is signal (right), meaning that the generated signal is inversely proportional to the amount of the analyte. Thus, if there is no analyte present, the sensor surface will be occupied only with antibodies and will produce maximum signal. For clarity, high analyte concentrations give low or no signal, due to that no ligands are left for binding. Indirect assay is generally used for detecting small molecules.

C: IgG detection in direct assay. Anti-bovine IgG antibody was immobilized onto CMD-3D sensor (carboxymethyl dextran). IgG signal was determined in as little as 2min injection time

D. Detection of cocaine from saliva in competitive assay. Cocaine-conjugate was immobilized onto a sensor and free cocaine molecules were detected from saliva samples at below 1ng/ml level.