Conventional SPR: Still the Best Choice for my Biosensing Needs
Updated: Jan 4, 2022
When it comes to real-time, label-free optical techniques for biosensing, environmental monitoring, clinical diagnostics, or elucidating protein-protein interactions, surface plasmon resonance (SPR) often comes to mind for most researchers. In general, SPR signals arise from a change in local refractive index due to the presence of a biomolecular interaction on a thin metallic surface, thus eliminating the need for labels, and the data are collected in real-time. Conventional SPR has been established for decades and its advantages will be highlighted in this blog. Localized SPR (LSPR), on the other hand, may be less familiar to some people. Its difference in instrumentation setup and potential challenges will be outlined below.
Strengths of Conventional SPR
Affinité's portable SPR instruments’ core technology (benchtop qSPR and portable P4SPR instruments) uses the Kretschmann configuration in which the incident light interacts with surface plasmons on a thin gold film that is coupled to a glass prism (Fig. 1). Upon generation of surface plasmons, evanescent waves propagate perpendicular to the metal surface into the surrounding sample media. These evanescent waves are sensitive to any refractive index changes within 200-300 nm from the thin metallic film surface . This is the key to the surface sensitivity of SPR - only changes in refractive index within this region would affect the SPR response. The presence of protein binding to surface receptors, for instance, would cause a change in the surface plasmon conditions, hence shifting the SPR response, which is measured in wavelength (and can be easily converted to resonance units) in our instruments.
Figure 1. Image of a conventional SPR setup based on the Kretschmann configuration.
What makes Affinité's instruments the go-to platform for most applications, quality control, and assay development? The gold film on the prism is ~50 nm thick, which has been shown to provide the most sensitive measurement . This is also the standard and preferred configuration with most SPR instruments on the market. Furthermore, our SPR instruments use wavelength mode, not angle. Thus, the optical components allow our platforms to stay compact and robust and less subjected to noise generated by physical vibration or perturbations. Most importantly, due to the refractometric nature of this technique, complex matrices such as serum and plasma can be used as the light does not go through the sample. Since conventional SPR emerged decades ago, there are already countless publications on the passivation of gold surfaces to minimize non-specific adsorption. Affinité has even patented a unique reagent called Afficoat to prevent fouling from complex biological samples. Most importantly, obtaining kinetic and affinity information for the biomolecular interaction of interest is very simple due to our easy-to-use SPR platform, intuitive software, and innovative KNX2 pump module.
The main advantages of conventional SPR for biosensing, assay development, and other applications include:
Minimal sample preparation
High sensitivity to surface refractive index changes induced by binding interactions at the surface
Many publications available on technique, surface chemistry, assay development, affinity and kinetics determination, and applications
Can be used for complex matrices such as clinical samples
What About LSPR?
Instead of a thin metallic film, LSPR uses round nanoparticles as well as other shapes such as nanodisks, nanorods, and nanotriangles  that are usually less than 100 nm in diameter, which are used either in solution or immobilized on a clear substrate. For immobilized LSPR, the SPR signal is dependent on the local refractive index of the dielectric medium. When the nanoparticles are excited by incident light, surface plasmons (or free electron oscillation) are confined within the boundaries of the nanoparticle (Fig. 2). LSPR uses transmission mode, in which a peak absorption wavelength shift would correspond to a change in the local refractive index on the nanoparticle surfaces.
Figure 2. Image of an LSPR setup.
Although LSPR seems like a straightforward process, there are some caveats in terms of signal reproducibility, sensitivity, and signal-to-noise. Sensor performance issues such as low result repeatability may be encountered due to SPR plasmon peak broadening, which may occur due to a number of physical nanoparticle processes (e.g. radiative damping), and a combination of inhomogeneity in size, shape, surface roughness, and material used for these nanoparticles [4, 5]. The density of immobilized proteins may vary if the nanoparticle surface is inconsistent with aggregates, which may affect sensitivity. Sensitivity can also be dictated by the nanofabrication process of metal nanoparticles on surfaces  as it relies on the the batch-to-batch reproducibility in providing consistent sensitivity of these materials, whereas it is much easier to reproduce thin metallic films for conventional SPR sensing . Furthermore, the noise in LSPR experiments is higher than traditional SPR [7, 8]. Those who opt to measure low concentrations of analytes (e.g. fM) by using fewer nanoparticles may run into signal-to-noise issues. It is known to decrease as fewer nanoparticles are used [7, 9].
Although the number of publications on LSPR has begun to increase since the early 2000s , there is still a gap in the optimization of surface chemistry of nanoparticles. For example, there must be a drive to discover strategies to minimize non-specific adsorption on nanoparticles and substrate surfaces and direct biofunctionalization to “hot spots” of nanostructures where sensitivity to refractive index can be maximized [6, 9]. This is especially true when it comes to complex matrices such as clinical samples. Furthermore, according to one author, there are certainly features that are unique to LSPR, but it is not yet practical . To learn more about SPR and LSPR, please read our SPR Technote.
Conventional SPR and LSPR provide signals through different pathways. However, the surface chemistry on planar thin films is more established and the SPR signal does not rely on the quality and/or behaviour of nanoparticles as in LSPR, which can easily diminish the sensor performance. The surface chemistry and synthesis of nanoparticles must be well controlled to maximize performance. It is certainly interesting to further explore LSPR phenomena and applications, but it is still not well established enough to obtain reliable data for (bio)sensing in clinical and environmental monitoring applications, studying protein-protein interactions, quality control, and assay development.
To summarize, conventional SPR instruments such as those by Affinité Instruments offer:
Reliable, repeatable, and high quality results due to the uniformity of planar gold thin films and robustness of our proprietary instrumentation design based on wavelength interrogation
Simple and user-friendly analysis of complex biological samples without much sample preparation
With all the above points in mind, Affinité's SPR instruments are based on conventional, or propagating, SPR because it is a more established and reliable method that can provide researchers with quality data without any uncertainties.
The Affinité Advantage
Affinité Instruments’ P4SPR™ is a very user-friendly, compact, and portable instrument. In addition, samples do not need much preparation and can be manually injected into the instrument. The P4SPR™, compared to a traditional immunoassay such as ELISA, provides fast, real-time affinity and/or kinetic data.
Simplicity - Fast training, fast results
Versatility - Pharmaceutical, biosensing, assay development applications
Economy - Affordable, accessible
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