Instruments, Optics and Fluidics
What the box around the prism has to do, and which design compromises you inherit when you choose one.
Three engineering problems#
The previous guide gave us a resonance condition. Turning that into an instrument that a biologist can use before lunch required solving three problems that have nothing to do with plasmons: how to read the angle, how to deliver liquid reproducibly, and how to hold the temperature still enough that the measurement means anything.
It is worth appreciating how much of SPR’s success is fluidics and thermal engineering rather than optics. The physics was settled in 1971 (Kretschmann, 1971). The first commercial instrument did not appear until 1990, and what took the intervening years was microfluidics, surface chemistry and thermostatting (Sjölander & Urbaniczky, 1991; Löfås & Johnsson, 1990).
Four ways to read the resonance#
The resonance condition can be interrogated by scanning any of the parameters in the resonance condition. Which one an instrument scans determines almost everything about its character.
| Approach | What is varied | Strength | Cost |
|---|---|---|---|
| Scanning angle | A mirror or rotating stage sweeps θ and the whole dip is recorded | Huge dynamic range; the dip shape itself is available as a diagnostic | Slower — the sweep takes time, limiting time resolution |
| Fan-shaped (convergent) beam | Nothing moves: a wedge of angles arrives at once and a diode array reads the whole dip | Fast, no moving parts, full dip every sample | Optical design is exacting; the angular range is fixed at manufacture |
| Fixed angle, intensity readout | Angle held on the steep flank of the dip; reflected intensity is the signal | Very fast, and compatible with a camera — hence imaging | Linear only over a small range; large shifts walk off the flank |
| Wavelength interrogation | Angle fixed, wavelength scanned; the resonant λ is tracked (Jorgenson & Yee, 1993; Homola, 1997) | Well suited to fibre and remote probes with no moving parts | Needs a spectrometer; dispersion of the metal complicates interpretation |
The fan-shaped beam deserves a note because it is what most modern flow-based instruments use. A converging beam contains a range of incidence angles simultaneously; after reflection those angles map onto positions on a linear photodiode array, so a single frame contains the whole reflectivity-versus-angle curve. There is no scan and nothing moves, which removes an entire category of mechanical drift (Jönsson et al., 1991).
Fluidics: the unglamorous half#
Two liquid-handling philosophies exist, and the choice has real consequences for the data.
- Flow cells. Buffer passes continuously over the surface in a channel typically 20–100 µm high. Sample is injected as a plug. Dissociation is measured under continuous wash, so released analyte is carried away and cannot rebind. This is what makes clean off-rate measurement possible, and it is the dominant design (Sjölander & Urbaniczky, 1991).
- Cuvettes. A stirred volume sits above the surface. Sample is added and mixed. Cuvettes use less sample and allow long equilibration, but during dissociation the released analyte remains in the cuvette and rebinds, so off-rates are systematically underestimated unless the volume is exchanged.
Within a flow cell, the height matters more than most users appreciate. Flow in these channels is firmly laminar — Reynolds numbers are of order 1 — so there is no turbulent mixing to bring analyte to the wall (Squires et al., 2008). Transport across the last few micrometres happens by diffusion alone, through what is effectively an unstirred layer. The consequences are taken up in kinetics and mass transport.
Temperature: why the specification is that tight#
The refractive index of water changes with temperature at roughly −1 × 10⁻⁴ per kelvin, which is large compared with the index change a small binding event produces (Jung et al., 1998). A binding event that produces 10 RU corresponds to a refractive index change of order 10⁻⁶. Setting those side by side: a temperature excursion of 0.01 K produces about the same signal as a 10 RU binding event.
That single comparison explains the entire thermal design of an SPR instrument — the massive thermostatted block, the pre-equilibration of buffer before it reaches the cell, the insistence on letting the system settle for an hour before real data. It also explains why a reference channel is not optional: temperature drift is common-mode and subtracts out, whereas nothing else will remove it (Myszka, 1999).
Instrument classes, without the product catalogue#
Specific models date quickly and vendors change hands; what persists is the class of design and the trade it embodies. Choose by the question you are asking.
| Class | Typical character | Best for | Weak at |
|---|---|---|---|
| Serial flow, few channels | 2–4 flow cells, one reference, fan-beam or scanning-angle readout | High-quality kinetics on a handful of interactions; the reference standard for a defensible k_a and k_d | Throughput |
| Parallel flow arrays | Crossed channel geometry giving a ligand × analyte grid in one run | Panels of tens of interactions with consistent conditions | Ultimate sensitivity; more complex referencing |
| SPR imaging | Fixed angle, camera readout, spotted arrays of 10²–10³ features (Rothenhäusler & Knoll, 1988; Jordan et al., 1997) | Epitope binning, multiplexed screening, array-format work (Abdiche et al., 2014) | Dynamic range; kinetic precision on any single spot (Berger et al., 1994) |
| Cuvette systems | Small stirred volume above the sensor | Precious samples; long equilibrations; equilibrium measurements | Off-rates, because released analyte rebinds |
| Localised SPR / nanoparticle | No prism; colour shift of a nanostructured surface (Haes & Duyne, 2002) | Compact, low-cost, point-of-care formats | Absolute quantitation; a much shallower sensing volume |
| Fibre-optic probes | Wavelength interrogation on a coated fibre (Jorgenson & Yee, 1993) | Remote and in-situ sensing | Referencing and temperature control |
Two neighbouring technologies are worth naming so that they are not mistaken for SPR. The resonant mirror, commercialised as IAsys, used an evanescent field in a waveguide structure rather than a plasmon (Cush et al., 1993). And one recurring source of confusion deserves clearing up. Biolayer interferometry instruments are not SPR instruments. They answer overlapping questions and their sensorgrams look similar, but they use interference between reflections at a fibre tip and involve no plasmon at all (Abdiche et al., 2008). related techniques treats them properly; the reason to mention it here is that "we measured binding by SPR on an Octet" is a sentence that appears in the literature and it is simply wrong.
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.
- Abdiche et al., 2014Y. N. Abdiche, K. C. Lindquist, A. Pinkerton, J. Pons, A. Rajpal (2014). Expanding the ProteOn XPR36 biosensor into a 36 × 36 array format provides a fast and economical means of determining antibody affinities and epitope bins. PLoS ONE 9, e92451. doi:10.1371/journal.pone.0092451 unverifiedHigh-throughput binning of a large antibody panel; the source for the n² scaling argument.
- 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.
- Cush et al., 1993R. Cush, J. M. Cronin, W. J. Stewart, C. H. Maule, J. Molloy, N. J. Goddard (1993). The resonant mirror: a novel optical biosensor for direct sensing of biomolecular interactions. Part I: Principle of operation and associated instrumentation. Biosensors and Bioelectronics 8, 347–354. unverified
- 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.
- Homola, 1997J. Homola (1997). On the sensitivity of surface plasmon resonance sensors with spectral interrogation. Sensors and Actuators B 41, 207–211. doi:10.1016/S0925-4005(97)80297-3 unverifiedQuantitative comparison of angular and spectral readout sensitivity.
- Jönsson et al., 1991U. Jönsson, L. Fägerstam, B. Ivarsson, B. Johnsson et al. (1991). Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. BioTechniques 11, 620–627. unverifiedThe paper describing the first commercial SPR biosensor as an integrated system — optics, microfluidics and sensor chip together. The primary source for what that instrument was and did.
- 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.
- Jorgenson & Yee, 1993R. C. Jorgenson, S. S. Yee (1993). A fiber-optic chemical sensor based on surface plasmon resonance. Sensors and Actuators B 12, 213–220. doi:10.1016/0925-4005(93)80021-3 unverifiedWavelength-interrogated SPR on an optical fibre, with no moving parts.
- 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.
- Kretschmann, 1971E. Kretschmann (1971). Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflächenplasmaschwingungen (The determination of the optical constants of metals by excitation of surface plasmons). Zeitschrift für Physik 241, 313–324. doi:10.1007/BF01395428 verifiedThe quantitative treatment: how dip position, depth and width relate to the metal’s dielectric function and film thickness.
- Löfås & Johnsson, 1990S. Löfås, B. Johnsson (1990). A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. Journal of the Chemical Society, Chemical Communications, 1526–1528. doi:10.1039/C39900001526 verifiedThe carboxymethyl dextran hydrogel — the single most consequential surface-chemistry paper in the field.
- Myszka, 1999D. G. Myszka (1999). Improving biosensor analysis. Journal of Molecular Recognition 12, 279–284. unverifiedOrigin of double referencing and blank-injection subtraction as standard practice.
- 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.
- Sjölander & Urbaniczky, 1991S. Sjölander, C. Urbaniczky (1991). Integrated fluid handling system for biomolecular interaction analysis. Analytical Chemistry 63, 2338–2345. doi:10.1021/ac00020a025 verifiedThe microfluidic cartridge that made SPR a routine instrument rather than a physics experiment.
- Squires et al., 2008T. M. Squires, R. J. Messinger, S. R. Manalis (2008). Making it stick: convection, reaction and diffusion in surface-based biosensors. Nature Biotechnology 26, 417–426. doi:10.1038/nbt1388 unverifiedThe general transport analysis for any surface-based sensor, in dimensionless form. The clearest statement of when a measured rate is chemistry and when it is delivery.