MACS Matchmaker
Regeneration prepares the same chip for another cycle, and it means one of two different things here. Which one applies is decided by how the ligand is attached, not by the interaction being measured.
- Regenerating the duplex — the normal case. On a DDI sensor the DNA anchor is dehybridized, which takes ligand and analyte off together, and a fresh ligand is loaded for the next cycle. Nothing has to survive the condition except the chip's capture oligo, so the same standard solutions work across interactions.
- Regenerating the ligand — stripping the analyte while the ligand stays attached and active. This is what other platforms usually mean by regeneration, and it is the harder problem: the condition must break the complex without denaturing the ligand, so it has to be found per interaction by scouting. On the MACS Matchmaker it is needed only where the ligand cannot be released — the covalently coupled [Me-Tz|PEG] and [Me-Tz|Tz] click sensors.
Recommended Conditions
These are duplex conditions: they dehybridize the DNA anchor on an [Oligo|PEG] or [Oligo|Oligo] surface, and both have been tested for it. The guanidinium solution can be ordered as the MACS Matchmaker Regeneration Solution from the Lino Biotech webshop.
- 50 mM NaOH
- 3 M guanidinium chloride (GuHCl), 125 mM NaOH
Add a regeneration step with the Regeneration phase template in the Advanced Planner, or let the guided experiment wizard place it from the regeneration solution you define. We generally recommend a short, fast pulse — 400 µL/min for 30 s — followed by a running-buffer rinse, and several cycles when a single pulse leaves the surface incompletely regenerated. The full timing and rinse sit in the Regeneration row of Recommended Flow Parameters.
Some ligands bind the surface so firmly that no regeneration solution removes them. For these, the guided wizard offers a Clean chip with trypsin at the end option: after the final cycle it injects trypsin for 50 min in full-loop mode to digest all protein on the sensor, ligand included, then runs the regeneration solution once more to flush the digest off. This regenerates the whole surface for the next immobilization. It does not replace the regeneration solution used between cycles.
Choosing Conditions by Bond Type
This table and the ladder below are for regenerating the ligand: the condition has to break the ligand–analyte complex and leave the ligand behind. Where the duplex can be dehybridized instead, neither is needed.
| Dominant interaction | Disruption strategy | Example conditions |
|---|---|---|
| Electrostatic / ionic | pH shift or high ionic strength | 10 mM Glycine-HCl pH 2.0, or 1 M NaCl |
| Hydrogen bonds | Chaotropic agents, low pH or high pH | 3 M GuHCl, 8 M Urea, or 50 mM NaOH |
| Hydrophobic | Mild surfactants | 0.5% SDS, or Tween 20 above the 0.05% running-buffer level |
| Mixed / unknown | Combination (cocktail) approach | 3 M GuHCl + 125 mM NaOH |
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:
- Mild: Low pH buffer (10 mM Glycine-HCl pH 2.0) or moderate salt (0.5 M NaCl). Try this first for most antibody–antigen interactions.
- Moderate: 50 mM NaOH or 3 M GuHCl. Effective for most protein–protein interactions.
- Strong: Combined 3 M GuHCl + 125 mM NaOH.
Verify regeneration efficiency by comparing the baseline level before and after treatment. Recovery above about 95% is the working target and recovery below about 80% indicates conditions that are too mild or a surface accumulating irreversibly bound material; treat both as working criteria for this platform rather than validated limits, and establish the acceptable window for your own assay.
Baseline recovery on its own does not show that the ligand still works. Confirm both: that the baseline returns, and that a repeated control analyte still gives its expected response. A baseline that comes back over a ligand that has been stripped or denatured is not successful regeneration.
DDI Chips: Regeneration via DNA Dehybridization
This is the route to prefer wherever the chip allows it. Dehybridizing the DNA anchor strands removes the entire ligand layer, and a fresh ligand is re-immobilized for the next cycle, so no analyte-specific ligand regeneration condition has to be developed at all. It is the recommended approach for MCK experiments on DDI chips. What it does not do is clean the instrument: surface fouling and the fluidic path still need their own verification.
Scouting Protocol
Scouting is how a ligand regeneration condition is found, and on the covalently coupled [Me-Tz|PEG] and [Me-Tz|Tz] click sensors there is no alternative to it: the ligand cannot be released, so reusing the chip means stripping the analyte off a ligand that has to survive the treatment. Scout it there, and anywhere a novel analyte–ligand pair has to be regenerated in place:
- Immobilize ligand and perform a single analyte injection.
- Inject the mildest regeneration condition.
- Check baseline return — if > 95%, the condition is sufficient.
- If baseline return is poor, repeat with a stronger condition.
- 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).