Sensor Surfaces and Immobilisation Chemistry

The stack, from glass upwards#

A sensor chip is four or five layers, each solving a different problem. Understanding what each layer is for makes the chemistry choices obvious rather than arbitrary.

The gold thickness is not a manufacturing convenience. Too thin and light tunnels straight through, so the resonance is shallow; too thick and the evanescent field from the glass side cannot reach the far interface, so coupling is weak. The optimum for gold in the red is around 45–50 nm (Kretschmann, 1971). You can watch this happen in the interactive reflectivity figure by sweeping the thickness slider.

Self-assembled monolayers#

Nuzzo and Allara found in 1983 that organic disulfides adsorb spontaneously onto gold to form ordered films (Nuzzo & Allara, 1983). Bain, Whitesides and colleagues then characterised alkanethiol monolayers systematically: how fast they form, how chain length affects packing, how the terminal group controls the surface’s properties (Bain et al., 1989).

The practical significance is that the terminal group of the thiol becomes the chemistry of the surface. A ω-hydroxy alkanethiol gives a hydrophilic surface for hydrogel attachment; a carboxyl terminus gives coupling handles directly; an oligo(ethylene glycol) terminus gives a surface that proteins do not stick to (Prime & Whitesides, 1991). That last discovery is the reason low-fouling biosensor surfaces exist at all, and the structure–property rules behind it have since been mapped across dozens of terminal chemistries (Ostuni et al., 2001).

Why a hydrogel, and why 100–200 nm#

Löfås and Johnsson attached carboxymethylated dextran to a thiol monolayer on gold in 1990 (Löfås & Johnsson, 1990). It is difficult to overstate how much of SPR’s subsequent success rests on that one design choice.

A flat two-dimensional surface has three problems that the hydrogel solves at once:

1. Capacity. A monolayer of protein on a flat surface is a hard ceiling. A 150 nm hydrated gel holds far more ligand, and — critically — holds it within the ~200–300 nm evanescent field, so the extra capacity is actually visible to the instrument.
2. Environment. Protein pressed against a hard hydrophobic surface tends to spread and denature. In a hydrated polymer mesh it is suspended in something much closer to bulk solution, and retains activity.
3. Fouling. A hydrated, neutral-to-anionic polymer layer is a poor substrate for non-specific protein adsorption.

The thickness is chosen against the physics set out earlier. Penetration depth into water is a couple of hundred nanometres, so a 100–200 nm matrix sits comfortably inside the sensing volume. A 1 µm gel would hold more ligand but most of it would be invisible — and worse, ligand at the top of such a gel would bind analyte that contributes nothing to the signal, which distorts kinetics in a way that is very hard to detect.

Pre-concentration: the step people skip#

Covalent coupling from a dilute protein solution would be hopelessly inefficient if the protein were merely diffusing past. It is not. The carboxymethyl dextran is polyanionic above about pH 3.5; a protein below its isoelectric point is net cationic. In low-ionic-strength buffer, the electrostatic attraction concentrates protein into the matrix by orders of magnitude before any covalent chemistry happens (Johnsson et al., 1991).

Two failure modes follow directly from the physics. If the buffer contains salt, the attraction is screened and nothing loads — so the coupling buffer must be low ionic strength, and the protein must be buffer-exchanged out of PBS first. If the protein’s pI is below about 4.5, the window between the dextran pKa and the pI has essentially closed, and amine coupling will be poor no matter what you do. For acidic proteins, move to a capture strategy (Löfås et al., 1995).

The coupling chemistries#

Amine coupling

The default. EDC activates surface carboxyls; NHS (or sulfo-NHS) converts the unstable O-acylisourea into a more stable succinimidyl ester; primary amines on the protein — lysine side chains and the N-terminus — displace NHS to form an amide (Grabarek & Gergely, 1990). Johnsson, Löfås and Lindquist established the protocol on dextran surfaces (Johnsson et al., 1991), and it is still the first thing anyone tries.

• Advantages. Works on almost any protein; stable amide bond; well characterised.
• Disadvantages. Random orientation, because coupling goes through whichever lysine reacts first. If a lysine sits in or near the binding site, that molecule is inactivated. Surface activity of 30–70% is typical (Johnsson et al., 1995), and the resulting site heterogeneity is exactly what going beyond a 1:1 model discusses.
• Do not use when the binding site is lysine-rich, or the protein’s pI is too low to pre-concentrate.

Thiol coupling

Couples through cysteine, either a native surface cysteine or an engineered one. Because a protein typically has far fewer free cysteines than lysines, orientation is much better controlled (Löfås et al., 1995). Engineering a single surface cysteine at a position away from the binding site is the cleanest covalent immobilisation available.

Aldehyde coupling

Oxidise carbohydrate — periodate on a glycoprotein’s glycans — to aldehydes, then couple to a hydrazide surface. Since glycans are usually distant from protein binding sites, this gives good orientation for glycoproteins specifically (Löfås et al., 1995).

Capture strategies

Rather than coupling the ligand covalently, immobilise a capture reagent covalently and let it hold the ligand non-covalently but tightly.

The reference surface deserves as much thought as the active one#

A reference channel is only useful if it differs from the active channel in exactly one respect: the presence of ligand. Every other property — charge, hydrophobicity, matrix density, activation history — should match.

1. Activate and deactivate. Run the full EDC/NHS activation and ethanolamine blocking on the reference channel, just without injecting ligand. This matches the surface chemistry (Grabarek & Gergely, 1990).
2. Consider an irrelevant protein. Immobilising a similarly sized, similarly charged protein that does not bind your analyte matches the charge and steric environment better than a bare deactivated surface. It also introduces its own risks, so pick something well characterised.
3. Check it. Look at the reference trace before subtraction, every time. A large signal there means non-specific binding is not being controlled and subtraction will not save you.

Sources cited on this page

Listed alphabetically. Each badge records whether the bibliographic record was confirmed against Crossref. unverified marks a real, deliberately chosen source whose volume and page numbers we have not yet machine-checked — it is not a comment on the science.

• Bain et al., 1989C. D. Bain, E. B. Troughton, Y.-T. Tao, J. Evall, G. M. Whitesides, R. G. Nuzzo (1989). Formation of monolayer films by the spontaneous assembly of organic thiols from solution onto gold. Journal of the American Chemical Society 111, 321–335. doi:10.1021/ja00183a049 verified
  The systematic study of alkanethiol SAM formation: kinetics, chain-length dependence, packing.
• Cooper et al., 1998M. A. Cooper, A. Try, J. Carroll, D. J. Ellar, D. H. Williams (1998). Surface plasmon resonance analysis at a supported lipid monolayer. Biochimica et Biophysica Acta — Biomembranes 1373, 101–111. doi:10.1016/S0005-2736(98)00091-1 unverified
  Lipid-capture surfaces for membrane-associated interactions.
• Grabarek & Gergely, 1990Z. Grabarek, J. Gergely (1990). Zero-length crosslinking procedure with the use of active esters. Analytical Biochemistry 185, 131–135. unverified
  The EDC/sulfo-NHS two-step chemistry underlying amine coupling.
• Green, 1975N. M. Green (1975). Avidin. Advances in Protein Chemistry 29, 85–133. unverifiedreview
  Source of the avidin–biotin affinity figures used to justify streptavidin capture surfaces.
• Johnsson et al., 1991B. Johnsson, S. Löfås, G. Lindquist (1991). Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors. Analytical Biochemistry 198, 268–277. doi:10.1016/0003-2697(91)90424-R corrected
  Note on this citation. This paper is widely cited with the page range 168–277, which is a transposition error that has propagated through several secondary sources. The Crossref record for this DOI gives 268–277, which is also what volume 198 of the journal actually contains. We use the corrected range.
  The EDC/NHS amine-coupling protocol, still the default immobilisation chemistry three decades later.
• Johnsson et al., 1995B. Johnsson, S. Löfås, G. Lindquist, Å. Edström, R.-M. Müller Hillgren, A. Hansson (1995). Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies. Journal of Molecular Recognition 8, 125–131. doi:10.1002/jmr.300080122 unverified
  Measures surface activity across coupling chemistries on the same antibody — the primary source for the claim that amine coupling inactivates a substantial fraction of ligand.
• Jönsson et al., 1991U. Jönsson, L. Fägerstam, B. Ivarsson, B. Johnsson et al. (1991). Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. BioTechniques 11, 620–627. unverified
  The paper describing the first commercial SPR biosensor as an integrated system — optics, microfluidics and sensor chip together. The primary source for what that instrument was and did.
• Kretschmann, 1971E. Kretschmann (1971). Die Bestimmung optischer Konstanten von Metallen durch Anregung von Oberflächenplasmaschwingungen (The determination of the optical constants of metals by excitation of surface plasmons). Zeitschrift für Physik 241, 313–324. doi:10.1007/BF01395428 verified
  The quantitative treatment: how dip position, depth and width relate to the metal’s dielectric function and film thickness.
• Lahiri et al., 1999J. Lahiri, L. Isaacs, J. Tien, G. M. Whitesides (1999). A strategy for the generation of surfaces presenting ligands on self-assembled monolayers: synthesis, characterization, and surface plasmon resonance studies. Analytical Chemistry 71, 777–790. doi:10.1021/ac980959t unverified
  Two-dimensional low-fouling surfaces as an alternative to hydrogels, characterised by SPR.
• Löfås & Johnsson, 1990S. Löfås, B. Johnsson (1990). A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. Journal of the Chemical Society, Chemical Communications, 1526–1528. doi:10.1039/C39900001526 verified
  The carboxymethyl dextran hydrogel — the single most consequential surface-chemistry paper in the field.
• Löfås et al., 1995S. Löfås, M. Malmqvist, I. Rönnberg, E. Stenberg, B. Liedberg, I. Lundström (1995). Methods for site controlled coupling to carboxymethyldextran surfaces in surface plasmon resonance sensors. Biosensors and Bioelectronics 10, 813–822. doi:10.1016/0956-5663(95)99220-F verified
  Thiol, aldehyde and other orientation-controlling couplings beyond plain amine chemistry.
• Nuzzo & Allara, 1983R. G. Nuzzo, D. L. Allara (1983). Adsorption of bifunctional organic disulfides on gold surfaces. Journal of the American Chemical Society 105, 4481–4483. doi:10.1021/ja00351a063 verified
  The discovery that sulfur-containing organics self-assemble into ordered monolayers on gold — the chemical foundation of every SPR chip.
• Ostuni et al., 2001E. Ostuni, R. G. Chapman, R. E. Holmlin, S. Takayama, G. M. Whitesides (2001). A survey of structure–property relationships of surfaces that resist the adsorption of protein. Langmuir 17, 5605–5620. doi:10.1021/la010384m unverified
  What makes a surface low-fouling, tested systematically across dozens of terminal chemistries.
• Prime & Whitesides, 1991K. L. Prime, G. M. Whitesides (1991). Self-assembled organic monolayers: model systems for studying adsorption of proteins at surfaces. Science 252, 1164–1167. doi:10.1126/science.252.5009.1164 verified
  Showed that oligo(ethylene glycol)-terminated monolayers resist protein adsorption — the origin of every low-fouling SPR surface.
• Raether, 1988H. Raether (1988). Surface Plasmons on Smooth and Rough Surfaces and on Gratings. Springer Tracts in Modern Physics, vol. 111. doi:10.1007/BFb0048317 unverifiedbook
  The standard monograph on surface plasmon physics, by one of the two people who first excited them optically.
• Schuck, 1996P. Schuck (1996). Kinetics of ligand binding to receptor immobilized in a polymer matrix, as detected with an evanescent wave biosensor. I. A computer simulation of the influence of mass transport. Biophysical Journal 70, 1230–1249. doi:10.1016/S0006-3495(96)79681-9 verified
• Sigal et al., 1996G. B. Sigal, C. Bamdad, A. Barberis, J. Strominger, G. M. Whitesides (1996). A self-assembled monolayer for the binding and study of histidine-tagged proteins by surface plasmon resonance. Analytical Chemistry 68, 490–497. doi:10.1021/ac9504023 verified
  NTA/Ni(II) capture of His-tagged protein on an SPR surface — the origin of the NTA chip.