SPR Imaging, LSPR, BLI and Interaction Networks
What sits next to SPR, how it differs, and where combining techniques is genuinely worth the effort.
SPR imaging#
Replace the single detector with a camera, hold the angle fixed on the flank of the dip, and every pixel becomes an independent refractive index sensor. Rothenhäusler and Knoll demonstrated the principle in 1988 (Rothenhäusler & Knoll, 1988); Corn and colleagues turned it into a quantitative tool for arrayed biological measurement (Jordan et al., 1997).
The trade is explicit and worth stating plainly.
- Gained: hundreds to thousands of simultaneous measurement spots, all under identical buffer, temperature and timing. For anything that scales as n² — binning above all — this is transformative.
- Lost: dynamic range and strict linearity, because intensity readout is only linear over a limited portion of the dip flank; and some kinetic precision on any individual spot, because camera noise exceeds photodiode noise.
Spot-to-spot variation is the practical headache (Berger et al., 1994). Every feature has its own local ligand density and its own baseline, so per-spot normalisation and generous internal referencing — reference spots distributed across the array rather than clustered at one edge — are essential rather than optional.
Localised SPR#
On a metal particle smaller than the wavelength of light, the plasmon cannot propagate. It resonates as a whole, and the resonance shows up as a strong extinction peak — the reason colloidal gold is red. Haes and Van Duyne turned this into a biosensor using triangular silver nanoparticles (Haes & Duyne, 2002).
A related hybrid worth knowing about is electrochemical SPR, in which the gold film doubles as a working electrode so that potential and binding can be controlled and observed together — an idea that goes back to Gordon and Ernst in 1980 (II & Ernst, 1980).
| Propagating SPR | Localised SPR | |
|---|---|---|
| Coupling | Prism, grating or waveguide required | None — direct illumination |
| Sensing depth | ~200–300 nm (Jung et al., 1998) | ~5–30 nm (Haes & Duyne, 2002) |
| Bulk refractive index sensitivity | High | Much lower |
| Relative sensitivity to material at the surface | Lower | Higher — the field is concentrated where the binding is |
| Instrumentation | Optical bench with angular or spectral readout | Can be a spectrophotometer, or the naked eye |
| Best at | Quantitative kinetics; absolute surface coverage | Compact, low-cost, point-of-care detection |
Biolayer interferometry#
BLI is the technique most often mistaken for SPR, so it is worth being precise. A biosensor tip carries two closely spaced reflecting interfaces. White light reflects from both; the two reflections interfere; binding at the outer surface changes its optical thickness and shifts the interference pattern. No plasmon is involved. Abdiche and colleagues characterised the platform against SPR in detail (Abdiche et al., 2008).
| SPR | BLI | |
|---|---|---|
| Physical principle | Surface plasmon resonance | Optical interference at a fibre tip |
| Sample handling | Flow cell; sample consumed continuously | Tip dips into a stirred well; sample recoverable |
| Dissociation | Under continuous wash — released analyte removed | Tip moves to a buffer well; rebinding is better controlled than a cuvette but less so than flow |
| Parallelism | Limited by flow channels | Trivially parallel — 8 to 96 tips at once |
| Noise | Lower | Higher (Abdiche et al., 2008) |
| Turbid samples | Tolerant — light never crosses the sample | Tolerant, for a different reason: sensing is confined to the tip |
| Best at | Precise kinetics; small analytes | Throughput; crude samples; quantitation |
Combining techniques: interaction networks#
The most interesting current use of these methods together is mapping networks of interactions rather than single pairs. A representative workflow, developed for protein–glycosaminoglycan interactions, runs like this:
- Discovery by SPR imaging. Array many candidate partners and screen a panel of analytes against all of them in parallel. This is a yes/no experiment and imaging is ideal for it.
- Confirmation and kinetics by flow SPR. Take the hits and characterise them properly on a low-density surface, where the constants mean something (Karlsson & Fält, 1997).
- Extension by BLI. Use the parallel format for the larger set of secondary interactions where throughput matters more than precision (Abdiche et al., 2008).
- Assembly into a network, annotated with affinities, and compared against curated interaction databases.
What makes this more than a list of pairwise measurements is that the affinities become edge weights. Whether a competing pair of partners matters biologically depends on their relative affinities and their relative abundances, and a network annotated with real kinetic constants supports that reasoning in a way a binary interaction map cannot.
Sources cited on this page
Listed alphabetically. Each badge records whether the bibliographic record was confirmed against Crossref. unverified marks a real, deliberately chosen source whose volume and page numbers we have not yet machine-checked — it is not a comment on the science.
- Abdiche et al., 2008Y. Abdiche, D. Malashock, A. Pinkerton, J. Pons (2008). Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet. Analytical Biochemistry 377, 209–217. unverifiedCharacterisation of biolayer interferometry against SPR.
- Berger et al., 1994C. E. H. Berger, R. P. H. Kooyman, J. Greve (1994). Resolution in surface plasmon microscopy. Review of Scientific Instruments 65, 2829–2836. doi:10.1063/1.1144627 unverifiedQuantifies the lateral resolution limit imposed by plasmon propagation length — the reason imaging spots cannot be made arbitrarily small.
- Haes & Duyne, 2002A. J. Haes, R. P. Van Duyne (2002). A nanoscale optical biosensor: sensitivity and selectivity of an approach based on the localized surface plasmon resonance spectroscopy of triangular silver nanoparticles. Journal of the American Chemical Society 124, 10596–10604. doi:10.1021/ja020393x verifiedThe localized SPR (LSPR) biosensor — no prism, no propagating wave, nanoparticle resonance instead.
- II & Ernst, 1980J. G. Gordon II, S. Ernst (1980). Surface plasmons as a probe of the electrochemical interface. Surface Science 101, 499–506. unverifiedThe origin of electrochemical SPR.
- Jordan et al., 1997C. E. Jordan, A. G. Frutos, A. J. Thiel, R. M. Corn (1997). Surface plasmon resonance imaging measurements of DNA hybridization adsorption and streptavidin/DNA multilayer formation at chemically modified gold surfaces. Analytical Chemistry 69, 4939–4947. doi:10.1021/ac9709763 verifiedSPR imaging applied to a real multiplexed biological measurement.
- Jung et al., 1998L. S. Jung, C. T. Campbell, T. M. Chinowsky, M. N. Mar, S. S. Yee (1998). Quantitative interpretation of the response of surface plasmon resonance sensors to adsorbed films. Langmuir 14, 5636–5648. doi:10.1021/la971228b unverifiedThe reference treatment of how a thin adsorbed film of known thickness and refractive index maps onto an SPR shift, including the exponential weighting of the evanescent field.
- Karlsson & Fält, 1997R. Karlsson, A. Fält (1997). Experimental design for kinetic analysis of protein–protein interactions with surface plasmon resonance biosensors. Journal of Immunological Methods 200, 121–133. doi:10.1016/S0022-1759(96)00195-0 verifiedWhere the low-density / high-flow-rate / analyte-range design rules come from.
- Rothenhäusler & Knoll, 1988B. Rothenhäusler, W. Knoll (1988). Surface–plasmon microscopy. Nature 332, 615–617. doi:10.1038/332615a0 verifiedFirst demonstration that the SPR signal can be imaged, not merely averaged over a spot.