Glossary
21 terms. Anything marked with a dotted underline in the text links here.
analyte
The partner flowed over the surface in solution.
The molecule injected in the mobile phase. Its molecular weight sets how much signal a binding event produces, which is why small-molecule work is harder than protein–protein work.
avidity
Apparent affinity enhancement from multivalent binding.
A bivalent analyte such as an IgG that engages two surface sites dissociates far more slowly than either arm alone, because both must release simultaneously. The fitted k_d is then not a property of the interaction but of the surface density. This is the most common reason antibody off-rates are reported as tighter than they are.
BLI
Biolayer interferometry — a fibre-optic label-free technique that dips into wells rather than flowing.
White light reflects from two interfaces on a biosensor tip and interferes; binding changes the optical thickness and shifts the interference pattern. Not SPR — no plasmon is involved — but it answers similar questions with different trade-offs: no fluidics, easy parallelisation, higher noise, and different sensitivity to sample turbidity.
bulk shift
The instantaneous square-wave jump caused by the sample buffer differing from running buffer.
Because SPR senses refractive index, any mismatch between sample and running buffer produces a step at the start of injection and an equal step down at the end. It is not binding: it appears instantly, has no curvature, and is identical on a reference surface. Subtracting a reference channel removes most of it; matching buffers removes the rest.
carboxymethyl dextran
The flexible carboxylated polysaccharide hydrogel that forms the standard SPR sensor surface.
A ~100–200 nm hydrated polymer layer carrying carboxyl groups for coupling. It suspends ligand in a solution-like environment rather than pressing it against a hard surface, provides a large binding capacity within the evanescent field, and resists non-specific protein adsorption. Its thickness was chosen to sit comfortably inside the ~200–300 nm sensing depth.
CFCA
Measuring active concentration from transport-limited initial rate, without a standard curve.
Deliberately operate under full mass transport limitation, where the initial binding rate depends only on the analyte’s diffusion coefficient, the flow-cell geometry and its concentration. Because none of those requires a standard of the same molecule, an absolute active concentration comes out. It measures *active* concentration, which is often the number you actually wanted and is usually lower than what a protein assay reports.
double referencing
Subtract a reference flow cell, then subtract a buffer-only injection.
The first subtraction removes bulk shift and non-specific binding common to both cells. The second removes systematic artefacts of the injection itself — valve switching spikes, small drift — that are present even with no analyte. Together they are the minimum acceptable processing for published kinetic data.
ka
Association rate constant, units M⁻¹ s⁻¹.
The second-order rate constant for complex formation. Diffusion sets a ceiling of roughly 10⁶–10⁷ M⁻¹ s⁻¹ for protein–protein association in the absence of electrostatic steering; values reported above ~10⁷ M⁻¹ s⁻¹ from a biosensor should be treated as suspect until mass transport has been ruled out.
kd
Dissociation rate constant, units s⁻¹.
The first-order rate constant for complex breakdown. Complex half-life is ln2/k_d. Very slow off-rates (below about 10⁻⁵ s⁻¹) cannot be measured reliably in a normal dissociation window because the decay is indistinguishable from baseline drift.
KD
Equilibrium dissociation constant, K_D = k_d / k_a. Units of concentration.
The analyte concentration at which half the ligand sites are occupied at equilibrium. Lower means tighter. Because it is a ratio of two rate constants, two interactions with identical K_D can have wildly different residence times — which is why reporting K_D alone throws away information you already paid to measure.
ligand
The partner immobilised on the sensor surface.
In SPR usage the ligand is whichever partner is attached to the chip, regardless of which one a pharmacologist would call the ligand. The convention is positional, not chemical, and it is a frequent source of confusion when talking to people outside the field.
LSPR
The non-propagating plasmon resonance of a metal nanoparticle.
On a particle smaller than the wavelength, the plasmon cannot propagate and instead resonates as a whole. No prism and no angle scan are needed — the resonance shows up as a colour change. The sensing volume is much smaller than for propagating SPR, giving different sensitivity to small analytes very close to the surface.
mass transport
When delivery of analyte to the surface, not chemistry, sets the observed rate.
Analyte must diffuse across the unstirred layer at the flow-cell wall before it can bind. If binding is fast and sites are plentiful, the surface consumes analyte faster than flow can resupply it, and the measured rate reflects diffusion rather than chemistry. The fitted k_a is then an underestimate and the curve takes on a characteristic near-linear association and a slow, non-exponential dissociation.
preconcentration
Electrostatic attraction of ligand into the dextran before covalent coupling.
Below its pI the ligand is net positive; the carboxylated dextran is net negative above about pH 3.5. Injecting ligand in low-ionic-strength buffer at a pH between the two concentrates it into the matrix by orders of magnitude, so covalent coupling proceeds efficiently from a dilute solution. Get the pH wrong and almost nothing immobilises.
regeneration
A short harsh pulse that strips bound analyte without destroying the ligand.
Usually 10–60 s of low pH (glycine pH 1.5–3.0), sometimes high salt, chaotrope or a pH jump upward. A good regeneration returns the baseline to its previous value and leaves binding capacity unchanged across cycles; a bad one degrades the surface a little each cycle, producing a decaying R_max that will masquerade as complex kinetics.
Req
The steady-state response reached during a long injection.
The plateau of the association phase, where association and dissociation balance. R_eq = R_max·C/(C + K_D), a rectangular hyperbola in analyte concentration — this is the basis of equilibrium (steady-state) affinity analysis.
Rmax
The response if every ligand site on the surface were occupied.
The saturation response of a surface. Predicted from the immobilised level scaled by the analyte-to-ligand molecular weight ratio and the stoichiometry. Comparing measured R_max with the prediction gives the surface activity, which is one of the most informative numbers you can report and one of the least often reported.
response units
Response unit: 10⁻⁴ degrees of resonance-angle shift, ≈ 1 pg mm⁻² of bound protein.
The instrument’s output unit. On Biacore-lineage instruments one RU is defined as a 10⁻⁴ degree shift of the resonance angle. Calibration against radiolabelled protein established the widely used equivalence of roughly 1 RU to 1 pg mm⁻² of adsorbed protein. The equivalence depends on the refractive index increment dn/dc of the adsorbing species and should not be applied unmodified to nucleic acids, lipids or heavily glycosylated proteins.
SAM
Self-assembled monolayer — an ordered organic film formed spontaneously on gold.
Alkanethiols chemisorb onto gold through the sulfur and pack into a crystalline-like film. The discovery that this happens spontaneously and reproducibly is what made designed sensor surfaces possible.
sensorgram
A plot of response against time — the raw output of an SPR experiment.
The response-versus-time trace for one flow cell. Reading sensorgrams by eye, before fitting anything, is the single most valuable skill in SPR.
SPRi
SPR read out as an image, so hundreds of spots are measured at once.
A camera replaces the single detector and the surface is illuminated at a fixed angle, so every pixel reports its own local refractive index. Trades some kinetic resolution for massive parallelism, which is what makes epitope binning of large antibody panels practical.