MACS Matchmaker
Every BIA experiment starts by attaching a ligand to the sensor surface. The choice of immobilization chemistry is not just a logistical detail — it shapes orientation, density, and stability, all of which feed back into the binding-model deviations covered in the BIA reference. A heterogeneous-ligand fit may simply be telling you that random amine coupling produced a mix of orientations; a mass-transport-limited fit may be telling you the surface density is too high. On lino, almost every chemistry is presented through a DNA duplex (DDI), which decouples the ligand-attachment chemistry from the chip itself — even covalent NHS, maleimide, and click captures are regenerable as long as the duplex is the weakest link. The exception is direct on-chip click on the [Me-Tz|PEG] / [Me-Tz|Tz] sensors, where the ligand is covalently bound to the ridges and the chip is single-use per ligand.
Pick a chip and adapter
Three quick questions — sample matrix, ligand type, and throughput — map onto a concrete sensor + adapter combination from the lino portfolio. Use this as a starting point, then refine density and orientation choices using the sections below.
Common methods at a glance
The table compares the most common immobilization chemistries by orientation control, reversibility, and where each shines.
| Method | Chemistry | Orientation | Reversible? | Best for | Protocol |
|---|---|---|---|---|---|
| On-chip amine coupling (NHS oligo adapter) | NHS-activated oligo hybridized first; covalent amide to surface –NH₂ on-chip | Random | Yes — dehybridize the duplex to release ligand+adapter | Unmodified proteins or peptides; no off-chip conjugation step | NHS-adapter on-chip protocol |
| Off-chip amine DDI (AminoLink kit) | Lysines conjugated to an oligo off-chip with the AminoLink kit, then hybridized | Random | Yes — strip the duplex between ligands | Reusable chip for multi-ligand panels; tighter control over conjugate stoichiometry | AminoLink DDI protocol |
| Off-chip thiol DDI (ThioLink kit) | Maleimide oligo coupled to a free / engineered cysteine, then hybridized | Semi-controlled | Yes — strip the duplex | Ligands with one accessible Cys; more reproducible orientation than amine coupling | ThioLink DDI protocol |
| DEL hit splint ligation (5′-phosphate adapter) | T4 DNA Ligase joins the DEL hit’s 5′-P tag to a chip-binding adapter via a splint oligo; the adapter then hybridizes onto the chip | DNA-tethered (compound at distal end of dsDNA tag) | Yes — strip the duplex | On-DNA DEL hit validation without re-synthesis; multiplex 16–54 compounds per chip | DEL splint-ligation protocol |
| Biotin capture (Anti-Biotin adapter) | Monovalent anti-biotin antibody — reversible capture without avidity-driven cross-linking, and the dissociation needed to keep backfilled grooves matched | Site-specific via biotin tag | Yes — duplex strip and mild antibody elution | Biotinylated ligands, including multi-biotinylated targets | Biotin capture protocol |
| Anti-tag capture (Protein A/G, trisNTA, Strep-Tactin XT, anti-GFP) | Capture ligand pre-loaded on the chip via its oligo adapter, then flow tagged ligand | Oriented | Yes — both via tag elution and via duplex strip | Tagged recombinants (His, Strep, GFP-fusion) and IgGs; reuse the chip across many ligands | Anti-tag capture protocol |
| Fc-receptor capture (FcγRs, FcRn) | Pre-conjugated FcγR / FcRn–oligo adapter (catalogue) hybridises to the chip; antibody flowed as analyte | Oriented (Fc-down) | Yes — duplex strip | Fc-effector profiling across FcγRI / IIa / IIb / IIIa / IIIb and IgG PK / half-life screening (FcRn) | Fc-receptor capture protocol |
| Off-chip TCO–tetrazine click (DDI) | TCO oligo adapter clicks to a tetrazine-tagged ligand, then hybridizes | Site-specific if tagged off-chip | Yes — duplex strip | Custom-tagged ligands where you still want a regenerable surface | TCO activation protocol |
| On-chip TCO–tetrazine click (Sensor [Me-Tz|PEG] / [Me-Tz|Tz]) | Bioorthogonal IEDDA between TCO-tagged ligand and Me-Tz ridges — direct, no DDI | Site-specific if tagged off-chip | No — chip is single-use per ligand | Long-lived covalent attachment; lowest-cost sensor (CHF 300–350) | TCO activation protocol |
Orientation matters for kinetics
Random covalent attachment (NHS lysine coupling, AminoLink DDI) exposes binding sites in a mix of orientations — some accessible, some occluded — and shows up as heterogeneous-ligand kinetics with two relaxation timescales. Oriented capture via the lino adapters (Protein A/G or Fc receptors for IgGs, trisNTA for His-tagged proteins, Strep-Tactin XT for Strep-tag II, anti-GFP VHH for GFP-fusions, Anti-Biotin for biotinylated ligands) gives cleaner, more homogeneous data because every ligand is presented the same way.
Density tradeoffs
High ligand density gives more signal but invites mass-transport limitation (analyte gets consumed faster than it diffuses in) and rebinding (released analyte reattaches before leaving the boundary layer). For kinetic measurements on typical proteins, aim for low density — roughly 50–200 RU on Biacore-style sensors, scaled up for small-molecule analytes that produce less signal per binding event. Equilibrium titrations tolerate higher density.
Reference: ridges vs grooves, not a separate channel
Unlike SPR or BLI, focal molography does not require a dedicated reference channel for blank subtraction. Each mologram is its own reference: ligand sits on the activated ridges, the grooves between them stay inert, and the diffractometric readout intrinsically rejects anything that does not bind in the coherent ridge pattern. Bulk refractive-index changes, injection artifacts, and non-specific binding all show up as incoherent background and do not contribute to the diffraction signal.
Where a no-ligand mologram is still useful is as a chemistry control — confirming the adapter itself (e.g. trisNTA, Protein A/G, Anti-Biotin) does not bind your analyte. This is a sanity check, not a load-bearing subtraction, and a single reference spot anywhere on the chip is enough.
On the matrix-tolerant Oligo | Oligo sensors the grooves are not left inert — they are backfilled with a chemically matched, non-active molecule so that abundant matrix proteins bind ridges and grooves equally and cancel in the coherent signal (see Backfilling and NSB suppression). Only use an adapter for backfilling if its capture is dissociable; with an irreversible capture (e.g. NeutrAvidin–biotin) the ridge and groove chemistries cannot be kept matched and the two regions mix. For biotinylated ligands this is why the dissociable Oligo Adapter Anti-Biotin is recommended over NeutrAvidin.
FM specifics: ridges, DDI, and click chemistry
A mologram has interleaved ligand-on and ligand-off ridges, so what matters is selective coupling to the activated ridges, not bulk surface coverage. lino achieves this in two ways. (1) DNA-Directed Immobilization (DDI): the chip carries a capture oligo on the ridges, and an oligo adapter brings the ligand in by hybridization — used for every Oligo|PEG and Oligo|Oligo sensor. (2) Direct click chemistry on the [Me-Tz|PEG] / [Me-Tz|Tz] sensors, where TCO-tagged ligands react bioorthogonally with the tetrazine-functionalised ridges via inverse-electron-demand Diels–Alder (TCO–Tz IEDDA), not the more common azide–alkyne click. TCO–Tz is copper-free, fast, and selective, but the bond is irreversible — the chip becomes single-use per ligand. Random orientation on the ridge surface still produces the heterogeneous-ligand pattern, so for kinetics use an oriented adapter (Protein A/G, trisNTA, Strep-Tactin XT, anti-GFP, Anti-Biotin) or a site-specifically TCO-tagged ligand whenever possible.