MACS Matchmaker

Assay Types

The questions the instrument answers, and for each one the route from planning an experiment to reading its result. Pick the assay type from the question you have, not from the data you happen to hold — the choice determines the injection design, and a design cannot be fixed afterwards by analysis. The table covers the experiment-plan types you build in the planner; it is not a list of every evaluation. Equilibrium affinity analysis and DEL hit validation both run on designs already in the table, and are described under Evaluations.

Assay	Answers	Planned with	Evaluated as
Scouting	Does this ligand immobilize, and does the analyte bind it?	Wizard → Scouting	Read from the sensorgram; no evaluation
SCK	How fast and how tightly does it bind? (k_on, k_off, K_D)	Wizard → Binding Kinetics & Affinity → SCK	Determine Kinetics
MCK	The same, when the surface can be regenerated	Wizard → Binding Kinetics & Affinity → MCK	Determine Kinetics
LOD	What is the lowest concentration distinguishable from blank?	Wizard → LOD	Limit of Detection
Concentration determination	How much analyte is in this unknown sample?	Wizard → Concentration Determination	Concentration Determination

Two routes to a plan

Four goals are offered in the guided experiment wizard, which asks for a goal, the instrument setup, the samples and the method, and writes a complete injection plan you can still edit afterwards. Its goals are Binding Kinetics & Affinity, with SCK and MCK options, LOD, Scouting, and Concentration Determination.

You can also build a plan in the Advanced Planner from the phase templates below, or edit a plan created by the wizard.

Scouting

What it is. A qualification run, not a measurement. It asks whether the ligand immobilizes at all, whether backfilling behaves, and whether the analyte binds — a yes/no answer bought cheaply before a full kinetic run commits the chip and the sample. Both backfilling and the analyte injection are optional, so the same phase serves an immobilization-only check.

Planning. Wizard → Scouting. Unlike the kinetics assays there is no phase template to add by hand — the wizard writes the cycles from the ligand and analyte tables. Ligands are grouped in table order and a new cycle starts whenever a placement reappears; with several analytes configured, every cycle injects them in table order. What it produces is ordinary immobilization, association and regeneration phases, editable afterwards like any other.

Analysis. None. Read the sensorgram directly: an immobilization step that rises and holds, and an analyte response above the reference channel. What to do when it does not is in Troubleshooting.

Single-Cycle Kinetics (SCK)

What it is. One continuous run in which analyte concentration increases injection by injection, with no regeneration between them. The surface is never reset, so each injection starts from the occupancy the previous one left. It consumes less sample and less time than MCK and needs no regeneration condition at all — which is what makes it the default for a ligand you cannot strip without damaging.

Planning. Wizard → Binding Kinetics & Affinity → SCK, or build the phase by hand from the Single Cycle Kinetics dialog, which generates the dilution series from one expected K_D. Aim the series at 0.1–10 × K_D, and give the final dissociation long enough to drop by at least 5% of the association plateau.

Analysis. Determine Kinetics. Because the surface is never reset, the response carries over between injections: accumulating occupancy raises the level each cycle starts from, and the fit models that. It does not license every rising baseline — drift from surface loss, adsorption or an incomplete wash looks similar and is not accounted for, so check that the rise is consistent with the concentrations injected.

Occupancy carries forward. Each injection is followed by the same short buffer step, but it is far too brief to clear the surface — so the next injection starts from what the previous one left and the trace climbs in a sawtooth rather than returning to baseline. The labels give each concentration as a multiple of K_D. Only the final buffer step is lengthened, and it has to run long enough to resolve k_off.

Multi-Cycle Kinetics (MCK)

What it is. One concentration per cycle, each cycle followed by regeneration that returns the surface to its starting state. Each concentration is therefore measured in a separate binding cycle that starts from a regenerated baseline — the classical design, and the more forgiving one to fit. Separate cycles are not independent replicates: they share the chip, the ligand preparation and usually the sample preparation — at the cost of needing a regeneration condition that strips the analyte without deactivating the ligand.

Planning. Wizard → Binding Kinetics & Affinity → MCK, or the Multi Cycle Kinetics phase. Establish the regeneration condition first — see Sensor Regeneration, whose scouting protocol exists precisely for this. On DDI chips the whole ligand layer can be dehybridized and reloaded instead, which sidesteps the problem.

Analysis. Determine Kinetics, which takes single- and multi-cycle designs alike: it splits the series into its cycles and fits them together.

As acquired

As evaluated

Every cycle starts clean. The same five concentrations as the single-cycle design, each injected on its own with the surface regenerated (orange) in between — that is the run as it is acquired, on the left. The evaluation aligns the cycles at injection start and fits them together, which is the form an MCK series is normally shown in and the reason it is the more forgiving design: every curve begins from the same baseline, so no cycle inherits the previous one's occupancy.

Limit of Detection (LOD)

What it is. The lowest analyte concentration reliably distinguishable from a blank. This software takes it as the concentration whose response exceeds the mean blank by 3.2 population standard deviations of the blanks, per mologram; the LOD evaluation gives the full calculation. It is a property of the whole assay, not of the instrument: the same reader gives a different LOD for a different ligand, matrix or association time.

Planning. Wizard → LOD, or the LOD assay phase. The blanks are the measurement: they set the σ the detection limit is built from, so the LOD phase requires at least three and more will estimate that σ better. Use the same flow rate for association and dissociation to keep the noise contribution even, and extend association to accumulate more bound mass if the floor needs to come down.

Analysis. Limit of Detection, which fits the lowest concentrations linearly and reports the detection limit against the blank distribution. Background on what sets the floor is under Limit of detection and sensitivity.

The blanks are the measurement. The detection limit is where the response line crosses 3.2 standard deviations of the blank distribution, so it is set as much by the scatter of the blanks as by the slope of the response. Running more blanks tightens σ and lowers the limit; a steeper response — longer association, more bound mass — moves the crossing left.

Concentration determination

What it is. Reading an unknown sample's concentration off a standard curve measured under matched conditions. It asks how much, not how fast or how tightly, and it is the one assay here whose answer depends on a second measurement — the calibration — being right.

Planning. Choose Concentration Determination in the guided experiment wizard and add each calibration standard and each unknown sample as its own analyte in Samples, setting each row to Calibration or Unknown. Method creates one Calibration Curve titration per calibration analyte and one Unknown Concentration titration per unknown, each preceded by its own Immobilization phase, with a Regeneration between consecutive titrations, so every titration starts from the same surface. Every analyte gets its own series from settings shared within its role. Tick Covalently immobilized ligand in Method when the ligand survives regeneration, and a Regeneration runs after each Immobilization as well; leave it clear for a DDI ligand, which that regeneration would strip off the surface. Initialization runs buffer at the start, and a final regeneration closes the run. Prepare the standards in the same matrix as the unknown. Aim for more than five standard concentrations that cover the expected unknown concentration. To reuse a curve measured earlier, set every analyte to Unknown: the run then measures no calibration curve, and its evaluation takes the calibration from that earlier measurement.

Analysis. Concentration Determination, which inverts the fitted curve per sensor and combines the per-dilution estimates. An unknown only quantifies where the curve still resolves it, so plan the standard range to bracket the samples rather than merely reach them. Each evaluation fits one calibration curve, and the Data step asks which one when the calibration measurement holds several. Every unknown in the evaluation is read off that one curve, so give each evaluation only the unknowns measured against its standard.

The answer is only as good as the curve. Standards measured in the same matrix define the calibration, and the unknown's response is mapped back through it — an interpolation, never an extrapolation. Two different things go wrong at the top. A response beyond the highest standard (shaded) gets no concentration at all: it is reported as out of range and contributes nothing. A response still inside the range but up on the flattening curve does return a value, but a small response error there becomes a large concentration error. Both are reasons to bracket the samples rather than merely reach them.

The phases a plan is built from

Whatever the wizard writes, the plan itself is a sequence of phases, and the same phase templates can be added by hand in the Advanced Planner. Five of them carry the assays above; the rest are the surface preparation and the plumbing around it. Scouting is not among them — the wizard writes it as ordinary immobilization, association and regeneration phases, so there is nothing to add by hand.

Phase	What it contributes
Immobilization	Loads the ligand onto the mologram, optionally followed by backfilling. Available only on a plan that includes immobilization.
Regeneration	Strips bound analyte so the next cycle starts clean — the phase MCK depends on. Conditions and scouting are under Sensor Regeneration.
Titration Series	A dilution series injected in one sweep. The general-purpose concentration ramp behind both kinetics phases, usable on its own for a dose–response.
Single Cycle Kinetics	The SCK assay above, generated from one expected K_D.
Multi Cycle Kinetics	The MCK assay above: one concentration per cycle with regeneration between.
Limit of Detection	The LOD assay above, blanks included.
Calibration Curve	The standards half of concentration determination — a titration of known concentrations in the sample matrix. Available only on a plan that includes immobilization.
Unknown Concentration	The samples half: the unknowns injected under conditions matched to the calibration curve. Available only on a plan that includes immobilization.
Custom Phase	An empty phase you fill with injection commands yourself, for anything the generators do not cover.
Template	Inserts a phase saved earlier, carrying its samples and analytes with it — the way a working design is reused across experiments.