MACS Matchmaker
The NHS-adapter strategy enables covalent, high-density immobilization of unmodified, primary-amine-bearing ligands directly on an Oligo|PEG mologram. Unlike off-chip DDI, no oligo conjugation kit is required — the NHS-equivalent reactive group is presented through a bifunctional linker that is loaded onto the chip in situ. The duplex underneath stays the weakest link, so the surface is regenerable between ligands by alkaline dehybridization.
From the catalogue
Order the Oligo Adapter NHS directly from the lino Biotech webshop:
- Oligo Adapter NHS — CHF 195, 110 µL stock at 2.6 µM in PBST, 10 immobilisations at 200 nM (130 µL/injection), pre-conjugated to your chosen Seq ID (00–16) for multiplexing on Oligo|PEG (8×8 default; 6×9 on request). Keep the TCO–PEG–TFP linker on hand separately and reconstitute it fresh in MES pH 6.0 before each use.
Strategy: a three-component adapter system
Coupling proceeds in three loading steps on an Oligo|PEG sensor:
- Hybridise the MeTz-oligo adapter onto the chip, presenting tetrazine groups on every active ridge.
- Click the bifunctional linker (TCO–PEG–TFP) at mildly acidic pH. The TCO end reacts with the surface tetrazine via inverse-electron-demand Diels–Alder (IEDDA); the TFP / NHS ester points outwards, ready to couple primary amines.
- Inject the ligand. Surface lysines of the ligand react with the exposed TFP / NHS ester to form a stable amide bond, locking the ligand covalently onto the activated ridges.
A short ethanolamine wash quenches any residual TFP / NHS groups before analyte injection, and an optional alkaline regeneration step removes non-covalently bound analyte for ligand reuse.
Protocol
Step 1 — Sensor
Use any Oligo|PEG sensor (1- to 64-plex). Multiplexed formats are supported.
Step 2 — Adapter hybridisation
| Parameter | Value |
|---|---|
| Reagent | MeTz-functionalised oligo adapter, complementary to the chip oligo |
| Concentration | 100 nM |
| Buffer | PBST (phosphate-buffered saline + 0.05% Tween 20) |
| Flow rate | 10 µL/min |
Step 3 — Linker activation (TCO–tetrazine click)
| Parameter | Value |
|---|---|
| Reagent | TCO–PEG5–TFP (bifunctional linker) |
| Linker chemistry preference | TFP > STP > NHS (slower hydrolysis = higher coupling yield) |
| Concentration | 20–50 µM (optimal); up to 100 µM |
| Buffer | MES pH 6.0 + 0.05% Tween 20 |
| Flow rate | 10 µL/min |
| Incubation | 5 min |
Step 4 — Passivation
| Parameter | Value |
|---|---|
| Reagent | Ethanolamine, 1 M, pH 8.5 |
| Flow rate | 10 µL/min |
| Incubation | 5 min |
| Purpose | Quenches unreacted TFP / NHS esters on the activated molograms |
Step 5 — Ligand immobilisation
| Parameter | Value |
|---|---|
| Ligand | Any primary-amine-bearing protein or peptide (IgG, enzymes, etc.) |
| Concentration | 0.1–0.5 µM |
| Buffer | HEPES pH 7.0 + 0.05% Tween 20 |
| Flow rate | 10 µL/min |
| Expected coverage | ~190 pg/mm² covalent immobilisation under benchmark conditions |
Step 6 — Analyte injection
| Parameter | Value |
|---|---|
| SCK association flow rate | 30 µL/min (6-ch) / 60 µL/min (3–4-ch) / 100 µL/min (1-ch) |
| SCK dissociation flow rate | 200 µL/min |
Step 7 — Optional regeneration
An alkaline wash removes non-covalently bound analyte while leaving the covalently attached ligand in place — the chip can be re-used with fresh analyte injections. For full ligand replacement, dehybridise the duplex underneath and start again from Step 2.
Optimal parameters at a glance
| Parameter | Optimal value |
|---|---|
| Adapter | MeTz-functionalised oligo (preferred over TCO-oligo) |
| Linker | TCO–PEG5–TFP |
| Linker concentration | 20–50 µM |
| Linker buffer | MES pH 6.0 + 0.05% Tween 20, 5 min |
| Passivation | 1 M ethanolamine pH 8.5, 5 min |
| Ligand concentration | 0.1–0.5 µM |
| Ligand buffer | HEPES pH 7.0 + 0.05% Tween 20 |
| On-chip flow rate (all loading steps) | 10 µL/min |
| SCK association flow rate | 30 µL/min (6-ch) / 60 µL/min (3–4-ch) / 100 µL/min (1-ch) |
| SCK dissociation flow rate | 200 µL/min |
| Tween 20 | 0.05% in linker, passivation, and regeneration buffers |
| Ionic strength | Low (PBST or MES-buffered saline) to enable surface pre-concentration |
Practical notes
Looking for the broader chip + adapter selection? Start from the interactive decision tree — the NHS on-chip lysine-coupling branch lands here.
Troubleshooting
Symptom: low ligand coverage, weak ligand signal
The most common failure mode of the NHS-adapter protocol is poor electrostatic pre-concentration of the ligand onto the negatively charged oligo molograms. Without pre-concentration the ligand never reaches the local concentration needed for the covalent coupling to win against TFP / NHS hydrolysis, and the ligand-injection step in the sensorgram barely rises above baseline.
The relevant parameter is the buffer pH relative to the ligand's isoelectric point (pI):
- pH < pI → ligand net positive → strong attraction to the oligo surface → pre-concentration works → good covalent coverage.
- pH ≈ pI → ligand near-neutral → marginal pre-concentration → weak coverage.
- pH > pI → ligand net negative → electrostatic repulsion → little or no coverage.
The default ligand buffer here is HEPES pH 7.0. That works well for basic ligands (pI > 7.5: most IgGs, lysozyme, cytochrome c, many growth factors). It fails for acidic ligands (pI < 6: BSA ≈ 4.7, transferrin ≈ 5.5, fetuin, many enzymes).
Fix: lower the coupling-buffer pH
Drop the ligand buffer pH to roughly pI − 0.5 to pI − 1. A pragmatic mapping:
| Ligand pI | Recommended ligand buffer |
|---|---|
| > 8 | HEPES pH 7.0 (default) |
| 6.5 – 8 | HEPES pH 6.5 or MES pH 6.5 |
| 5 – 6.5 | MES pH 5.5 |
| < 5 | Sodium acetate pH 4.5 – 5.0 |
Symptom: ligand pI is unknown
Run a quick pH scout: inject the ligand briefly (after the MeTz-oligo and linker steps) onto a sensor in three or four buffers spanning pH 4.5 / 5.5 / 6.5 / 7.0. The buffer that gives the strongest non-covalent accumulation in real time is the right one for the actual coupling run. The ligand can usually be recovered by alkaline regeneration of the duplex if the scout uses a fresh sensor each pH.
Symptom: coverage is OK but background drifts upward
Most often a sign that the ligand or analyte is sticking to the PEG grooves rather than the activated ridges. Make sure 0.05% Tween 20 is present in every buffer (linker, ligand, passivation, regeneration), and consider switching to an Oligo|Oligo sensor for matrix work — the chemically defined grooves resist non-specific adsorption better than PEG.
Symptom: ligand precipitates near its pI
Aggregation close to the pI is common. Stay one pH unit away from the pI (above or below) and accept the lower coupling yield, or switch to off-chip conjugation with the AminoLink kit, where the ligand stays in a controlled, dilute buffer throughout the chemistry.
Symptom: ligand is regenerated off the surface during alkaline wash
The covalent amide bond is alkali-stable; the duplex underneath is what the regeneration is supposed to break. If the ligand comes off too easily, the coupling probably never went covalent — most often because the linker step was too short, the linker stock had hydrolysed, or the ligand was net-negative at the coupling pH (see the pI section above). Re-prepare a fresh linker semi-stock and re-check the buffer pH first.