While selecting a surface plasmon resonance (SPR) instrument, the biggest concern for the customer is its sensitivity and detection limit. The sensitivity of SPR is complex as there is no single term to define it. We will discuss some of the commonly used terms of SPR. The motive here is to provide the users of SPR with guidelines to determine sensitivity and detection limit if a certain definition is useful for a customer’s application.
The first term we will define is Angular Sensitivity. In angular sensitivity, the angle of incident light at which surface plasmon resonance takes place is measured. Depending on molecular binding incident onto the sensor surface or some kind of change in the refractive index (index refraction) of the medium near the sensor surface, the angular shift of the resonance defines the sensitivity. In this case, the minimum detectable angular shift is used to define sensitivity. This sensitivity also depends on the prism material, the dielectric constant of the metal and dielectrics, as well as on the wavelength of the light used to excite the surface plasmons.
The penetration of the optical signal in the medium depends on the upon the wavelength of the optical radiation and the penetration in the medium increases with the wavelength. For Longer wavelengths such as near-infrared, have the advantage of being able to investigate further beyond the surface of the sensor. This activity however results in a significant loss of surface sensitivity.
Another common term is the Relative Index of Refraction Unit (RIU). In contrast to the angular shift, the unit RIU is more significant to applications that demand an exact measurement of the index of refraction of a medium. For applications aspiring to study molecular binding events, RIU is not the best way to define. There can likely be a relationship between angular shift and RIU if one knows the exact instrumental conditions such as the wavelength of incident light and prism material. Note that an SPR instrument with the best sensitivity in terms of RIU does not always mean that it has the best sensitivity in terms of detecting molecular binding.
Surface Coverage can be used to detect molecular binding that takes place on the sensor surface. In this case, the appropriate way to define the sensitivity is in pg/mm. The unit of Response (RU) is defined as 1 RU= 1 pg/mm which is frequently used to determine surface coverage.
However, like other examples, this is not a universal definition. For example, sensitivity based on the size, optical polarizability and density of the molecules bound to the surface, may be different from an SPR measurement with respect to the mass per unit surface area. The polarizability depends on the wavelength of light, particularly when the wavelength is close to the optical absorption band of the molecules like UV-vis labels, chromosomes etc. As most of the proteins have analogous polarizabilities, the SPR signal may be considered approximately proportional to the coverage of molecules bound to the sensor surface, and pg/mm is a useful way to quantify SPR sensitivity.
Sensitivity is sometimes defined in terms of lowest detectable molar concentration however; a highly sensitive instrument cannot accurately guarantee the detection of an extremely low analyte concentration. Just because a sensor is highly sensitive doesn’t mean it is suitable for every application. This is because the detection limit and sensitivity are two different analytical “figures of merit”, which are frequently mixed. The instrumental noise in the background has some effect on determining the lowest detection level. Some of the factors that determine sensitivity are as follows:
- Molar concentration
- Molecular sizes. For example, those with small molecular weight and polarizability can be can be detected easily.
- Surface coverage and affinity of the captured molecules
- Operating temperature,
- Buffer solution and
- The thickness of the modifier layer and its refractive index.
- SPR binding responses such as binding assays, labels, enzymatic reactions, etc.
Hence, sensitivity of SPR in terms of lowest detectable molar concentration can be misleading and incredibly challenging to beginner SPR users.
Next, let’s discuss how detection levels are determined. There are many ways to determine Detection Levels as the definition of “lowest detectable level” is not distinctly signified. Some indicate the root-mean-square or standard deviation while others choose to use the peak-to-peak value of the noise in the SPR signal. In analytical chemistry, the most used definition of detection limit is three times the standard deviation of the background noise.
Though time-consuming, the noise can be filtered and by smoothening of data and time averaging, one can remove certain noises and improve both detection level and the sensitivity.
The noise level can also be influenced by electronic amplification. An increase of gain/amplification may improve the signal to noise ratio, but this typically affects the detection range or dynamic range of the instrument. Finally, when comparing imaging SPR or other pixel-based detectors, the sensitivity is determined by how many pixels the SPR signal is averaged over time. The more the pixels, the better the sensitivity, however this increased sensitivity comes at the cost of spatial resolution and response time.