Choosing Ligand and Analyte

The decision of which molecule to immobilize on the mologram (ligand) and which to flow in solution (analyte) affects data quality and interpretation. Consider the following guidelines:

• Immobilize the smaller, more stable molecule. A smaller ligand leaves more room for the larger analyte to bind without steric interference. Additionally, larger analytes produce a stronger signal per binding event (more pg/mm² per molecule), improving the signal-to-noise ratio.
• Flow bivalent molecules as analyte with caution. Bivalent analytes (e.g., full IgG antibodies) can bind two adjacent ligands simultaneously, introducing avidity effects that complicate kinetic analysis. If avidity is a concern, consider using monovalent fragments (Fab) as the analyte, or reduce ligand density to minimize the probability of bivalent binding.
• Preserve ligand activity. The immobilized molecule must remain functional after coupling. For click chemistry chips, covalent attachment via TCO-Tz/MeTz may affect protein folding if the label is placed near the binding site. For DDI chips, the DNA conjugation strategy is generally milder and more predictable.
• Practical constraints. Immobilize the molecule that is available in smaller quantity (less material is needed for surface immobilization than for flowing multiple concentrations). Flow the molecule that is easy to prepare in a dilution series.

Assay Phases

Each assay type starts from a phase template. Use the guidance below for phase-specific design choices; for all flow rates and injection volumes, see Recommended Flow Parameters.

Baseline

Allows the system to stabilize before the first relevant injection. Run the running buffer until the baseline is flat.

Association & Immobilization

• Frontfilling and backfilling can be configured with the Immobilization template when appropriate (see Immobilizations).
• In the guided experiment wizard, choosing the [oligo | oligo] sensor architecture adds an empty ligand row to the Samples step with its placement set to Backfilling.
• For titration experiments (LOD, SCK, MCK, …) the sample/target must be defined as the Analyte.
• To tune ligand surface density on DDI chips, see Ligand Density vs. Mass Transport Trade-Off.

Kinetics Injections

• Use a kinetics phase template when it matches your experiment.
• Typical durations: 2 min association, 2 min dissociation.

Scouting

Use Scouting to confirm that your assay works before committing instrument time to a full experiment: that the ligands immobilize, that backfilling behaves as expected, and that the analyte binds. It also gives a quick yes/no answer on whether a ligand binds its analyte. Backfilling and analyte injection are both optional. When multiple analytes are configured, every Scouting cycle injects them in table order, using the same association and dissociation sequence for each.

Ligands are grouped in table order. A new Scouting cycle starts whenever any placement, including backfilling, appears again. When Inject as Pool is enabled, the non-backfilling ligands in each cycle are combined into one injection.

Scouting cycles omit leading run-buffer commands from immobilization, association, and regeneration phases when the preceding phase already ends with run buffer. Repeated Immobilization, Scouting, and Regeneration phases receive numbered names so that every phase name is unique.

Limit of Detection (LOD)

Experimental setup for LOD measurements. For analysis parameters and the LOD calculation method, see LOD Evaluation.

• Use an LOD-oriented phase template if available.
• Blanks: > 5 times.
• Typical durations: 2 min baseline, 5 min association.
• Flush sample after association: 200 µL flush at 200 µL/min.
• Use the same flow rate for association and dissociation to minimize noise impact.

Concentration Determination

• Use a concentration-determination phase template if available.
• Standards: > 5 different concentrations, duplicates recommended.

Experiment Planning

Injection Commands

Inject and Incubate

When selecting the pickup volume for the inject and incubate command, consider the effects of diffusion from the autosampler loop. As illustrated in the figure below, a larger volume reduces the impact of diffusion. Both the incubation duration and fluid viscosity influence this process. Given a sensor volume of 3 µL in the single flow chamber, a default value of 30 µL is recommended.

Inject with Alternate Flow

If the sample volume is limited and/or a high flow rate is required, the alternate flow command can be used. This pushes and pulls the sample over the sensor for a specified time. The pickup volume is the amount of sample that is delivered to the sensor. The displacement volume defines how much the sample is pushed forth and back. Both parameters can be set in the command dialog. Large displacement volumes lead to loss of the sample after a few minutes due to the dilution and diffusion effects. A displacement volume of less than 10 µL is recommended to avoid excessive diffusion. For an incubation of more than 5 minutes, a displacement volume of 1-2 µL is recommended.

Stop Condition

Each command includes an optional Stop Condition that automatically terminates the command based on specific criteria. There are three selectable stop conditions:

1. None: Default setting. The command runs until completion unless manually edited or fast-forwarded.
2. Plateau: Monitors the median coherent mass density of active signal molograms. The system records 10 initial data points, then evaluates whether the last 4 points deviate less than 1% from their median. When this criterion is met, the command fast-forwards.
3. Level Reached: Terminates when the median coherent mass density reaches a configured target. Three sub-options select how the target is interpreted:
   • ↑ Rise (association): the value entered is a delta (Δ); the target is starting signal + Δ. The command stops when the last 2 points (in pg/mm²) are all at or above the target.
   • ↓ Fall (dissociation): the value entered is a delta (Δ); the target is starting signal − Δ. The command stops when the last 2 points are all at or below the target.
   • ≷ Absolute value (auto-detect direction): the value entered is treated as an absolute target in pg/mm² rather than a delta. The direction is inferred from the signal at the start of the command: if the initial 4-sample window sits below the target the command stops on an upcross; if it sits above, the command stops on a downcross.

   Examples (Δ-based): if the baseline is 40 pg/mm² and you enter a magnitude of 25 with 'Rise', the command stops once the median reaches 65 pg/mm². With 'Fall' it would stop at 15 pg/mm².

   Example (absolute): with an 'Absolute value' target of 5 pg/mm² and an initial signal of ~40 pg/mm² (higher than the target), the command stops when the signal falls to 5 pg/mm² — useful as a safety floor that prevents, e.g., zero-crossing.

Autosampler Injection Modes

The MACS Sampler supports three injection modes. The software automatically selects the best mode based on the programmed injection volume:

RSD = Relative Standard Deviation, a measure of injection reproducibility (lower is better).

Samples and Buffer Recommendations

• Focal molography is more tolerant of sample-to-running-buffer mismatch than SPR, so exact matching is not always required.
• For quantitative kinetics and whenever bulk effects are visible, still minimize mismatch between running buffer and sample buffer.
• Suitable buffers: PBS, HEPES, or Tris-based systems. All buffers should be supplemented with surfactant molecules, such as Tween 20, at a recommended concentration of 0.05% v/v.
• Buffers and samples can also contain pre-blocking molecules, such as BSA, casein, or MoloBlock.
• For complex media samples, we recommend centrifuging samples and using only supernatant to prevent clogging of the tubing with aggregated sample.
• Avoid particulate matter in samples to prevent clogging of the fluidic system.

Recommended Flow Parameters

The MACS Matchmaker is compatible with aqueous solutions from pH 1–13. For flow chamber layouts and mologram counts, see Chip Assembly.

Default values: use the preferred-flow column as the starting point for new experiments, then adjust if sample volume, kinetics, mass transport, or regeneration strength require it.

Injection volume:use > 90 µL (avoid < 50 µL) to prevent sample dilution from tail diffusion.

Effect of Flow Rate on Association Kinetics

The overlay below shows the same association phase at different flow rates. If the sensogram shape changes when you increase the flow rate, mass transport is likely limiting your measurement — continue increasing until the curves overlay.

Measuring in complex media

Complex media — serum, plasma, cell lysate, conditioned media, crude supernatant — carry high-abundance background proteins in the µM–mM range. Focal molography is built for these samples: coherent non-specific binding is rejected by affinity-matched backfilling, and the ridge–groove self-reference cancels the bulk and thermal terms that swamp refractometric sensors. As a result you can usually measure in undiluted matrix, without the dilution, washing, and proofreading steps a refractometric assay needs. The guidelines below turn that capability into reliable numbers.

1. Start with a backfilled chip

Use an affinity-matched (backfilled) sensor: [oligo | oligo] for regenerable DDI assays or [MeTz | Tz] for covalent click chemistry. A passivated [… | PEG] chip is only appropriate in buffer. Pick the product from the Chip Selection Guide; for custom ligands, design the backfill to the criteria under Choosing a backfill probe.

2. Prepare the sample

• Clarify:centrifuge and use only the supernatant; filter through 0.2 µm where the sample allows. Particles must stay below 50 µm to avoid clogging the fluidics.
• Keep it cold:hold samples at 4 °C in the MACS Sampler to limit degradation and protease activity during long titrations.
• Mind viscosity: plasma and serum are more viscous than buffer, which thickens the diffusion boundary layer and can push the assay toward mass-transport limitation — account for it when interpreting on-rates.

3. Buffer and blocking

Run a buffer that matches the sample’s ionic strength and pH, with 0.05% Tween 20 to suppress non-specific adsorption and air bubbles. For serum or plasma, add a blocker such as 0.1% BSA (or casein / MoloBlock) to the buffer and, where appropriate, the sample. See Samples and Buffer Recommendations.

4. Run the right controls

• Blank matrix: inject the sample background without analyte and confirm the CMD baseline stays flat. A rising CMD on a matrix-only injection signals a ridge–groove mismatch (see Verifying backfill quality). The MD channel is expected to rise as the surface fouls — that is normal.
• Spike recovery: spike a known amount of analyte into the matrix and into buffer; comparing the responses quantifies matrix effects on binding and detection.
• Reference molograms: keep non-binding reference molograms in the layout for an additional, in-matrix subtraction channel.

5. Calibrate in the matrix

For concentration determination, build the standard curve in the same matrix as the unknowns. The specific signal is preserved in complex media, but the calibration slope can drop modestly relative to buffer because of transport and competition — so a matrix-matched standard curve gives accurate back-calculated concentrations. See Concentration Prediction.

6. Regenerate and clean

Complex samples foul the surface even when the coherent signal stays clean, so cleaning is not optional. Regenerate between cycles as needed (Regeneration Conditions) and run the cleaning protocol after complex-sample runs (Maintenance & Troubleshooting). For quantifying the smallest detectable concentration in matrix, see Limit of detection and sensitivity.

Recommended Regeneration Conditions

Regeneration in focal molography refers to the process of removing bound analytes from the mologram surface to allow for chip reuse or sequential measurements. The regeneration strategy must be tailored to the ligand–analyte interaction and the chemical robustness of the surface. The following regeneration solutions have been tested and work for regenerating [oligo|PEG] or [oligo|oligo] surfaces:

• 50 mM NaOH
• 3 M Guanidinium Chloride, 125 mM NaOH

For the standard regeneration timing and follow-up rinse, see the Regeneration row in Recommended Flow Parameters. Multiple regeneration cycles can be applied when incomplete surface regeneration is observed. When sticky proteins cannot be regenerated from the surfaces, incubate with 1× trypsin/EDTA followed by the regeneration solutions above (full-loop injection of 500 µL trypsin at 10 µL/min for 50 min).

Choosing Regeneration Conditions by Bond Type

Escalation Strategy

Always start with the mildest effective condition and escalate only as needed. Harsh regeneration risks denaturing the ligand or damaging the antifouling coating on the chip surface:

1. Mild: Low pH buffer (Glycine-HCl pH 2.5) or moderate salt (0.5 M NaCl). Try this first for most antibody–antigen interactions.
2. Moderate: 50 mM NaOH or 3 M GdnCl. Effective for most protein–protein interactions.
3. Strong: Combined 3 M GdnCl + 125 mM NaOH, or enzymatic digestion with trypsin/EDTA for very sticky analytes.

Verify regeneration efficiency by comparing the baseline level before and after treatment. Recovery of > 95% indicates complete regeneration. Recovery < 80% suggests the conditions are too mild or the surface is accumulating irreversibly bound material.

DDI Chips: Regeneration via DNA Dehybridization

DDI chips offer a distinct advantage: the entire ligand layer can be removed by dehybridizing the DNA anchor strands (e.g., with 50 mM NaOH), and a fresh ligand can be re-immobilized for the next cycle. This eliminates the need to develop analyte-specific regeneration conditions and is the recommended approach for MCK experiments on DDI chips.

Regeneration Scouting Protocol

When developing regeneration conditions for click chemistry chips or novel analyte–ligand pairs:

1. Immobilize ligand and perform a single analyte injection.
2. Inject the mildest regeneration condition.
3. Check baseline return — if > 95%, the condition is sufficient.
4. If baseline return is poor, repeat with a stronger condition.
5. After finding an effective condition, verify that repeated regeneration cycles (5–10×) do not reduce ligand activity (i.e., the binding capacity should remain stable across cycles).