NTA–modified Sensor Chips

XanTec NiD and NiHC sensor chips are coated with a bioinert (poly)carboxylate hydrogel matrix that has been modified with nitrilotriacetic acid (NTA) functionalities. These sensor chips allow the reversible capture immobilization of His-tagged biomolecules through complex formation with transition metal ions, preferably Ni2+. For sufficient binding stability, the His-tag should contain six to ten histidine residues. For complete regeneration of NTA-modified sensor chips, the NTA(Ni2+)-His-tag complex can be cleaved by chelating agents such as EDTA or imidazole.

Binding Characteristics:

NiD Sensor Chips: The binding affinity of Ni2+-loaded NiD sensor chips is primarily governed by the typical monovalent interaction between NTA(Ni2+) and His-tags, with dissociation rates (koff) in the range of 10-3 s-1.

NiHC Sensor Chips: Multivalent binding character, leading to a significant increase in binding stability of up to three orders of magnitude. Baselines after His-tagged ligand capture show minimal to no drift, with typical koff values of  10-5–10-6 s-1.1

Key features:

For further details, find a list of the most popular XanTec NTA sensor chips below. 

Product code2 NiP NiD200M NiHC200M NiHC1500M
Base Coating 2D, ultra-short bioinert CM-dextran (high density) 3D, 200 nm bioinert CM-dextran (medium density) 3D, 200 nm bioinert polycarboxylate (medium density) 3D, 1500 nm bioinert polycarboxylate (medium density)
Typical specific binding 
capacity [µRIU]3 		≈ 100 ≈ 400 ≈ 1200 ≈ 2000
Ligands His-tagged peptides and proteins
Analytes
  • Proteins
  • Nucleic acids
  • Carbohydrates
  • Viruses and cells
  • Proteins
  • Peptides
  • Nucleic acids
  • Nucleic acids
  • Small molecules
  • Peptides
  • Carbohydrates
  • Nucleic acids
  • Small molecules
  • Small carbohydrates
Intended purpose
  • Works best in combination with EDC/NHS coupling for ligand stabilization 
  • Well-suited for studying protein-protein interactions
  • Optimized for low-density immobilization with minimal nonspecific binding
  • Equivalent to competitor NTA chips
  • Suitable for weak binders
  • Works best in combination with EDC/NHS coupling for ligand stabilization
  • Ideal for medium to small analytes
  • Suitable for situations which require higher capture densities
  • Suitable for the analysis of weak and strong binders
  • Optimized for the analysis of small analytes
  • Ideal for applications requiring maximum capture capacities
  • Suitable for the analysis of weak and strong binders 

1 High binding stability requires immobilization at approximately one-third of the chip’s maximum capacity
2 Table includes a selection from XanTec’s full NTA sensor chip portfolio
3 Binding capacities are based on the capture level of His₆-peptide from a 5 µM solution after a 180 s stabilization period. Immobilization capacities of His-tagged proteins are considerably higher.

PAG–modified Sensor Chips

XanTec PAGP, PAGD, and PAGHC sensor chips are coated with a bioinert polycarboxylate matrix, pre-functionalized with recombinant 50.5 kDa Protein AG. This non-glycosylated Protein AG variant includes four IgG-binding domains derived of Protein A and two IgG binding domains from Protein G, providing high-affinity binding to the Fc region of all human IgG subclasses. Additionally, it exhibits strong affinity for mouse IgG2a, IgG2b, and IgG3, as well as total IgG from other mammals, including cow, goat, sheep, and rabbit. 

To ensure highly specific IgG binding, non-essential regions—such as the cell wall binding region, cell membrane binding region, and albumin-binding domain—have been removed from the recombinant Protein AG.

XanTec’s Protein AG sensor chips are ready-to-use, eliminating the need for time-intensive assay optimization and streamlining workflows. With five distinct Protein AG-modified versions available for SPR applications, each is tailored to specific analytical needs:

Key features:

For further information, refer to the table below, which highlights some of XanTec’s most widely used PAG sensor chips:

Product code1 PAGP PAGD200L PAGHC30M PAGHC200M
Base Coating 2D, ultra-short bioinert CM-dextran (high density) 3D, 200 nm bioinert CM-dextran (low density) 3D, 30 nm bioinert polycarboxylate (medium density) 3D, 200 nm bioinert polycarboxylate (medium density)
Typical specific binding 
capacity [µRIU]2 		≈ 5,000 ≈ 12,000 ≈ 6,000 ≈ 11,000
Ligands Fc region of all human IgG subclasses, strong affinity for mouse IgG2a, IgG2b, and IgG3, total IgG from cow, goat, sheep, and rabbit
Analytes
  • Proteins
  • Peptides
  • Nucleic acids
  • Proteins
  • Peptides
  • Nucleic acids
  • Small molecules
  • Proteins
  • Peptides
  • Nucleic acids
  • Carbohydrates
  • Proteins
  • Peptides
  • Nucleic acids
  • Carbohydrates
  • Small molecules
Intended purpose
  • Capture immobilization of Fc fusion proteins and antibodies for the analysis of medium- to large-sized analytes
  • Capture immobilization of Fc fusion proteins and antibodies for the analysis of small- to medium-sized analytes
  • Antibody quantification 
  • Capture immobilization of Fc fusion proteins and antibodies for the analysis of medium- to large-sized analytes including carbohydrates
  • Capture immobilization of Fc fusion proteins and antibodies for analysis of small- to medium-sized analytes including carbohydrates
  • Antibody quantification

1 Table includes a selection from XanTec’s full PAG sensor chip portfolio.
2 Maximum binding capacities are determined based on the capture level from a 100 µg/mL solution of Protein A–purified, pooled human IgG in PBS buffer.

Streptavidin–modified Sensor Chips

XanTec streptavidin-modified sensor chips are coated with a bioinert (poly)carboxylate matrix, pre-functionalized with a recombinant 52 kDa streptavidin tetramer. This immobilized streptavidin efficiently captures biotinylated biomolecules—including proteins, nucleic acids, peptides, and other biotin-tagged ligands—under physiological conditions. Thanks to the exceptionally high binding affinity of streptavidin for biotin (dissociation equilibrium constant ≈ 10-15 M), XanTec’s SA sensor chips enable the stable immobilization of biotinylated ligands with negligible dissociation.

Key features:

For further details, find a list of the most popular XanTec SA sensor chips below.

Product code1 SAP SAD200M SAD700L SAHC30M SAHC200M SAHC1500M
Base coating 2D, ultra-short CM-Dextran
(high density)		 3D, 200 nm bioinert CM-dextran
(medium density)		 3D, 700 nm bioinert CM-dextran
(low density) 		3D, 30 nm bioinert polycarboxylate
(medium density) 		3D, 200 nm bioinert polycarboxylate
(medium density) 		3D, 1500 nm bioinert polycarboxylate
(medium density)
Specific binding capacity [µRIU]2 ≈ 600–1200 ≈ 4000–5000 ≈ 6000–8000 ≈ 1200–1800 ≈ 3500–5000 ≈ 4500–6000
Recommended ligands Biotinylated
  • Proteins
  • Peptides
  • Nucleic acids
 Biotinylated
  • Proteins
  • Peptides
  • Nucleic acids
  • Carbohydrates
Recommended analytes
  • Proteins
  • Peptides
  • Nucleic acids
  • Viruses and cells
  • Proteins
  • Peptides
  • Nucleic acids
  • Small molecules
  • Peptides
  • Nucleic acids
  • Small molecules
  • Proteins
  • Peptides
  • Nucleic acids
  • Carbohydrates
  • Proteins
  • Peptides
  • Nucleic acids
  • Small molecules
  • Carbohydrates
  • Peptides
  • Nucleic acids
  • Small molecules
  • Small carbohydrates
Intended purpose
  • Kinetics of medium and large analytes
  • Especially suitable for weak binders with fast on- and off-rates
  • Capture immobilization of biotinylated secondary antibodies
  • All-purpose
  • Kinetics of medium and small analytes
  • Competitors SA sensor chip equivalent
  • Especially suitable for situations which require high capture densities
  • Kinetics of small analytes
  • Equilibrium analysis
  • Especially suitable for situations which require very high capture densities
  • Kinetics of medium and large analytes,
  • Especially suitable for weak binders with fast on- and off-rates including carbohydrates
  • Capture immobilization of biotinylated secondary antibodies
  • All-purpose
  • Kinetics of medium and small analytes including carbohydrates
  • Especially suitable for situations which require high capture densities
  • Kinetics of small analytes, including carbohydrates
  • Equilibrium analysis
  • Especially suitable for situations which require very high capture densities

1 This overview represents a selection of the full streptavidin sensor chip portfolio.
2 Based on the specific binding of 100 µg/mL biotinylated BSA in PBS.

HCX Sensor Chips (NHS–preactivated)

XanTec’s HCX sensor chips are based on a 3D hydrogel matrix composed of flexible polycarboxylate chains grafted onto a hydrophilic adhesion promoter on a gold support. The carboxylate groups are partially pre-activated with NHS esters, providing medium to high activation densities and enabling high immobilization capacities. This pre-activation significantly simplifies and accelerates ligand coupling, making HCX chips a powerful tool for streamlined workflows. They are particularly advantageous for spot immobilization in imaging SPR systems.

Though a certain fraction of the spotted ligand will immobilize when drying the spots, electrostatic preconcentration substantially increases immobilization yields. A preconcentration scouting step is therefore recommended prior to coupling. Small molecules, however, freely diffuse into the hydrogel and do not require preconcentration for efficient immobilization.

Key features:

For further details, find a list of XanTec HCX sensor chips below.

Product code1 HCX30M HCX200M HCX1000M
Base Coating 3D, 30 nm bioinert polycarboxylate
(medium density)
NHS pre-activated		 3D, 200 nm bioinert polycarboxylate
(medium density)
NHS pre-activated		 3D, 1000 nm bioinert polycarboxylate
(medium density)
NHS pre-activated
Specific binding capacity [µRIU]2 ≈ 9000 ≈ 15000 ≈ 20000
Recommended ligands
  • Proteins
  • Peptides
  • Nucleic acids
  • Small molecules
  • Proteins
  • Peptides
  • Nucleic acids
  • Proteins
  • Peptides
  • Nucleic acids
Recommended analytes
  • Proteins
  • Peptides
  • Nucleic acids
  • Viruses and cells
  • Small molecules (limited)
  • Carbohydrates
  • Peptides
  • Small molecules
  • Nucleic acids (low molecular weight)
  • Carbohydrates (low molecular weight)
  • Small molecules
  • Fragments
  • Carbohydrates (low molecular weight)
  • Nucleic acids (low molecular weight)
Intended purpose
  • Kinetics of medium and large analytes including carbohydrates
  • Especially suitable for weak binders with fast on- and off-rates
  • Immobilization of secondary antibodies and other capture molecules
  • Kinetics of small analytes
  • Equilibrium analysis
  • Especially suitable for situations which require high capture densities
  • Immobilization of secondary antibodies and other capture molecules
  • Equilibrium analysis
  • Fragment screening
  • Concentration analysis
  • Especially suitable for situations which require very high capture densities
  • Immobilization of small capture molecules

1 This overview represents a selection of the full HCX sensor chip portfolio.
2 Based on the specific covalent immobilization of 100 µg/mL BSA in 5 mM sodium acetate pH 5.0.

Agarose

Agarose (MW approx. 120 kDa) monolayer grafted to a thin adhesion promoter.

Linear polymer consisting of alternating D-galactose and 3,6-anhydro-L-galactopyranose linked by α-(1→3) and β-(1→4) glycosidic bonds.

Alginate

Alginate (or alginic acid) is a biopolymer that has found numerous applications in biomedical science and engineering. Alginate hydrogels are particularly attractive in wound healing, drug delivery, and tissue engineering applications, as these gels retain structural similarity to the extracellular matrices in tissues.

Alginate is a linear polymer composed of (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G), covalently linked together in different sequences. The monomers can appear in homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M-blocks), or alternating M and G-residues (MG-blocks). Alginates are refined from brown seaweeds of the class Phaeophyceae. As a natural product, alginates from different species of seaweed have variations in their chemical structure, resulting in different physical properties.

Click chemistry (AZC, AZD, DCD)

Click Chemistry or Click Coupling has gained increasing popularity among chemists because of its outstanding reaction kinetics, excellent selectivity, and superior stability in physiological environments. To make this bioconjugation technology available for ligand immobilization in SPR biosensing, polycarboxylate sensor coatings are modified with a very small, bioinert, azido (N3) group, while the ligand is labeled with a low-molecular-weight cyclooctyne linker (DBCO), a procedure comparable to ligand biotinylation. These groups can react in a quantitative and highly-selective manner via a simple alkyne–azide cycloaddition, forming a stable, covalent triazole linkage.

Some expression systems are capable of site-specific integration of non-natural amino acids containing an azide moiety. For efficient, selective and site-directed Click Coupling of such ligands XanTec also offers DBCO-modified sensor chips.

You can find more information about azide- and DBCO-modified polycarboxylate sensor chips for Click Chemistry here.

Linear polycarboxylate (C)

The robust C hydrogel consists of a hydrophilic, strictly linear polycarboxylate with a polyalkyl backbone. Compared with CM-Dextran (CMD) coatings, this hydrogel shows significantly lower diffusion limitation, resulting from its small molecular footprint and higher flexibility of the polymer chains. It has a high carboxyl group density and is a good choice if the ligand and analyte are small and high immobilization density is required. Due to the high negative charge density, the C coating is particularly well suited for acidic analytes, such as DNA. Harsh conditions are tolerated as the C–C backbone withstands practically all aqueous buffers and organic solvents except strong oxidizing agents.

Chondroitin sulfate (CDS)

CDS is an unbranched glycosaminoglycan composed of a chain of alternating sugars (N-acetyl-D-galactosamine and D-glucuronic acid) where each sugar can be sulfated in variable positions and quantities.

As a mucopolysaccharide and component of cartilage tissue, CDS shows good biocompatibility while being mechanically relatively rigid. The abundant carboxyl groups allow for activation using standard EDC/NHS chemistry.

CM-Cellulose

Carboxymethyl cellulose (CM-Cellulose, CMC) is a cellulose derivative with carboxymethyl groups (–CH2–COOH) bound to hydroxyl groups of the glucopyranose monomers that make up the cellulose backbone. Dependent on the degree of carboxymethylation and the polymer length, functional properties of CMC vary. CMC sensor surfaces can be useful for interaction studies of plant derived biomolecules or cellulose-related research.

Carboxymethyldextran (CMD)

Dextran and its derivative CM-Dextran have a long history as biocompatible materials and the crosslinked form of CMD (Sephadex®) has been used for more than 50 years as a stationary phase for chromatography of biomolecules. These polysaccharides are relatively bioinert and moderate flexibility of the polymer chains gives immobilized ligands a degree of freedom within the hydrogel. Dextran-based hydrogel coatings thus provide a good environment for many biomolecular interactions. The polymer main chain consists of glucose monomers with α-1,6 glycosidic linkages and occasional branches formed by α-1,3 forks. Carboxymethylation—usually one COOH per anhydroglucose unit—introduces negatively charged carboxyl groups into the dextran, which can be activated with EDC/NHS and allow for a range of secondary derivatizations, such as amino, thiol or azide groups.

CM-Dextran coatings also serve as the basis for further sensor chip types or modifications, such as Protein A/G for capture of antibodies, NTA for His6-tags, streptavidin for biotinylated proteins, and lipophilic anchors for immobilization of vesicles and micelles.

Hydrogels from CM-Dextran coatings are suitable for many applications. However, in interaction measurements with other carbohydrates or with highly glycosylated proteins, nonspecific interactions can be high, making CMD less suitable for such analytes/ligands. Furthermore, carbohydrate-based polymers are quite bulky, occupy a significant fraction of the evanescent field, and can cause diffusion artifacts.

Planar carboxymethyldextran (CMDP)

Short carboxymethyldextran (CMD) chains in train conformation, i.e. attached almost flat to a thin adhesion promoter layer. The dextran matrix of this <5 nm thin composite coating has the same molar carboxymethylation level as our 3-dimensional CMD sensor chips, i.e. one carboxymethyl group carried per anhydroglucose unit.

The hydrated yet thin structure of this coating effectively suppresses nonspecific interactions while avoiding steric effects, which is advantageous when working with large molecules (MW >200 kDa). The CMDP coating is also suitable for particulate analytes, such as viruses and whole cells. As for all carboxyl functionalized coatings, this surface is compatible with standard EDC/NHS immobilization chemistry.

Carboxymethylpolyethyleneglycol (CMPEG)

Carboxyl-terminated polyethylene glycol (4 kDa) terminally grafted to a hydrophilic adhesion promoter. Low non-specific binding and limited negative charge density (only one COO- functionality per PEG molecule). Immobilization of biomolecules via, e.g., EDC/NHS chemistry familiar from more common chip coatings like carboxymethyldextran (CMD).

Low-molecular-weight PEG coatings combine good entropic stabilization with the advantage that the interactions take place relatively close to the gold surface, i.e., in the most sensitive part of the evanescent field. This surface is an alternative for interaction analysis of medium- or high-MW analytes if dextran- or polycarboxylate-based surfaces cannot be used.

Sulfonated polycarboxylate hydrogel (CS)

The partially sulfonated “C“-matrix consists of a biocompatible, hydrophilic, strictly linear polycarboxylate comprising of a brush-structured polyalkyl polymer backbone in which some carboxylic groups have been replaced by more acidic sulfonates. Having a lower pKa compared with carboxylates, these sulfonate groups allow preconcentration of proteins with pI <4.25. The typical characteristics of the C matrix, such as reduced diffusion limitation for bigger molecules and higher flexibility of the polymer chains compared to polysaccharide-based hydrogels, are further advantages of these coatings.

Dextran (D)

Coatings of brush-structured, unmodified dextran (D) are relatively bioinert and electrostatically neutral. The flexibility of the unbranched polymer chains gives immobilized ligands a degree of freedom. Dextran-based hydrogels thus provide an excellent environment for many biomolecular interactions. The polymer main chain consists of α-1,6 glycosidic linkages between glucose monomers, with branches from α-1,3 linkages. In contrast to the usually employed carboxymethyl dextran (CMD), the dextran coating has no charge. Due to the lack of carboxylic groups, D coatings are not compatible with standard EDC/NHS immobilization chemistry. Alternative covalent immobilization methods include oxidation to polyaldehydes and subsequent Schiff base coupling/reductive amination or (bis)epoxy activation. As there will not be any electrostatic preconcentration, high ligand concentrations are necessary for sufficient immobilization.

Gelatin

Gelatin is an irreversibly hydrolyzed form of collagen with a broad molecular weight distribution of the polymer strands. Gelatin contains 19 amino acids, predominantly glycine, proline and hydroxyproline (>50%).

HC linear polycarboxylate

To minimize steric hindrance, diffusion artifacts and nonspecific binding to carbohydrate motifs, XanTec has made efforts to substitute polysaccharides such as CM-Dextran with a better defined polycarboxylate with excellent bioinertness and smaller molecular footprint compared with the relatively bulky saccharides. The linear, unbranched, highly hydrated polymer chains of the HC hydrogel are extremely flexible; thus, they give immobilized ligands a high degree of freedom and the ability to interact in a cooperative manner with larger analytes.

In addition to these biophysical advantages, the HC coatings contain no hydroxyl groups. Therefore, they cannot form ester crosslinks upon EDC/NHS activation, a common side reaction with carboxymethylated carbohydrates. The HC polymers are fully compatible with all immobilization chemistries available for CM-Dextran sensor chips.

Structural differences between surface-grafted hydrogels based on polysaccharide (left) and HC polycarboxylate (right).

Heparin (HEP)

Heparin is a carbohydrate of the glycosaminoglycan family and consists of variably sulfated repeating disaccharide units. The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated, N-sulfated glucosamine. Heparin as a sensor chip coating exhibits good bioinertness and can be useful in hematology research as it binds selectively plasma coagulation proteins, nucleic acid enzymes, lipases, and other proteins with specific heparin binding sites like lipoproteins and matrisome-associated proteins (e.g. growth factors and cytokines).

Hydrophobic planar alkyl layer

Dodecanethiol self-assembled monolayer. Contact angle >100°.

This flat, hydrophobic sensor surface facilitates adsorption of lipid monolayers for analysis of interactions involving lipid components. Liposomes adsorb directly onto the sensor surface and form a lipid monolayer with “tails” directed toward the hydrophobic alkyl layer and hydrophilic “heads” directed toward the solution. Unlike the usual strategy for immobilization of biomolecules, this surface has no functional groups suitable for forming covalent bonds.

Hyaluronic acid (HY)

yaluronic acid is a strongly hydrated, anionic, nonsulfated glycosaminoglycan, which makes it unique among glycosaminoglycans as they are usually sulfated. The molecular weight of the hyaluronic acid used for the HY coatings lies between 2 and 4 MDa, which corresponds to 15,000–25,000 disaccharide monomers. The hyaluronic acid used is of animal origin (chicken).

Hyaluronic acid is a polymer of disaccharides, themselves composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked via alternating β-(1→4) and β-(1→3) glycosidic bonds. Due to its high biocompatibility and common presence in the extracellular matrix of tissues, hyaluronic acid has been used as the basis for biomaterial scaffolds in tissue engineering.

As hyaluronic acid (HY) hydrogels are bioinert they can be a good choice for use in experiments with ligands/analytes that are prone to nonspecific interactions with CMD and HC surfaces. Further, they can be useful for biocompatibility research and tissue engineering.

Due to the abundant carboxyl groups, HY coatings are fully compatible with all immobilization chemistries available for CM-Dextran sensor chips.

Hydrazide modified linear polycarboxylate (HZHC)

Based on the linear polycarboxylate HC with its superior biocompatibility and diffusion properties, the hydrazide modification of this surface coating offers mild and covalent immobilization of oxidized (poly)saccharides, antibodies through their oxidized carbohydrate side chains, or other molecules with aldehyde or ketone functional groups. The resulting hydrazone bond is stable if formed from ketones; if an aldehyde is used, it is recommended to stabilize the double bond by reduction.

Lipophilic anchors (LD)

The 3D hydrogel LD surface—based on 500-nm carboxymethyl dextran coating—has been optimized to capture whole vesicles and liposomes. Covalently attached to a hydrophilic polymer, lipophilic alkyl chains act as “anchors” for particulates containing lipid membranes.

Planar lipophilic anchors (LP)

On the 2D LP surface, lipophilic alkyl chains are linked to a planar carboxymethyl dextran layer (CMDP surface). In contrast to the hydrophobic alkyl SAM-modified surface (HPP) that immobilizes lipid monolayers, the LP chip is the ideal substrate to prepare supported lipid bilayers. Such 2D structures can be simply prepared by vesicle spreading and mimic the cell membrane. LD and LP surfaces are suitable for analysis of membrane protein–protein interactions or membrane protein–inhibitor interactions.

NTA immobilized on CMD (NiD)

Nitrilotriacetic acid (NTA)-derivatized carboxymethyldextran allows immobilization of polyhistidine (His6)-tagged molecules via Ni2+/NTA chelation. Obvious advantages of this immobilization method are the site-directed, homogenous immobilization of ligands; capture in physiological conditions without significant changes of ionic strength and/or pH; and the ability to regenerate the sensor chip using chelators such as EDTA (350 mM) or imidazole (50 mM) followed by Ni2+ re-loading and immobilization of fresh ligand.

Although this approach is quite popular, a common problem with NTA sensor chips based on carboxymethyldextran is constant leaching of bound ligand from the chip surface, which results in a drifting baseline. If surfaces with a stable baseline are required, we recommend the use of our high-affinity poly-NTA sensor chips.

The use of 50 µM EDTA in running and sample buffers is recommended, as it decreases negative effects caused by contaminating heavy metal ions. Low concentrations of surfactant help to decrease nonspecific binding. HEPES buffer gives better results than Tris or PBS.

Multidentate poly-nitrilotriacetic acid (NTA)-derivatized linear polycarboxylate (NiHC)

NTA-derivatized carboxymethyldextran (CMD) allows immobilization of polyhistidine (His6)-tagged molecules via Ni2+/NTA chelation. Obvious advantages of this immobilization method are the site-directed, homogenous immobilization of ligands; capture in physiological conditions without significant changes of ionic strength and pH; and the ability to regenerate the sensor chip using chelators such as EDTA (350 mM) or imidazole (50 mM) followed by Ni2+ re-loading and immobilization of fresh ligand.

A common problem of NTA-derivatized CMD surfaces is the constant leaching of captured ligand from the chip surface, which results in a drifting baseline. Such drift effects can easily exceed the specific signal and thus represent a major problem, especially when screening small molecules. To address this unwanted effect, XanTec has developed a poly-NTA sensor chip hydrogel coating (HC) based on a flexible polycarboxylate polymer backbone. Compared with the standard CMD–NTA chemistry, these hydrogels, which are available in 30-, 200-, 1000-, and 1500-nm thickness, improve the immobilization stability of captured His6-tagged ligands by 2–3 orders of magnitude (Fig. 1). The higher stability of the bond results from a cooperative effect of several, closely spaced NTA groups.

Therefore, when selecting a sensor chip for immobilization of His6-tagged proteins in critical applications with small molecule analytes, the HC variant should always be preferred to the CMD-based chip. However, the increased density of NTA groups in the HC variant may lead to higher non-specific binding (NSB). Thus, if the analyte is a large protein, or the sample matrix is complex (e.g., it contains serum proteins), the choice should be a CMD-based NTA chip to avoid NSB.

Figure 1. Interaction/affinity map of His6-tagged A/G fusion protein with mono-NTA-derivatized carboxymethyl dextran (top) and XanTec’s poly-NTA sensor chip (bottom; NiHC1000M). Affinity increases from the upper-right corner to the lower-left corner. The affinity map was calculated from association/dissociation data using EvilFit5.
  1. Nieba, L., Nieba-Axmann, S. E., Persson, A., Hämäläinen, M., Edebratt, F., Hansson, A., ... & Plückthun, A. (1997). BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Analytical biochemistry, 252(2), 217-228.
  2. Oshannessy, D. J., Odonnell, K. C., Martin, J., & Brighamburke, M. (1995). Detection and quantitation of hexa-histidine-tagged recombinant proteins on western blots and by a surface plasmon resonance biosensor technique. Analytical biochemistry, 229(1), 119-124.
  3. Gorshkova, I. I., Svitel, J., Razjouyan, F., & Schuck, P. (2008). Bayesian analysis of heterogeneity in the distribution of binding properties of immobilized surface sites. Langmuir, 24(20), 11577-11586.

Protein A/G on carboxymethyldextran sensor chips

Recombinant fusion protein A/G (Mw around 50 kDa) is pre-immobilized either on a planar surface or in a 3-dimensional hydrogel consisting of carboxymethyldextranCMD). Recombinant protein A/G on these sensor chips consists of seven IgG-binding domains (EDABC–C2C3); the protein A portion (segments E, D, A, B, and C) is from Staphylococcus aureus, and the protein G portion (segments C2 and C3) is a Streptococcal cell wall protein. Sensor surfaces with protein A/G are designed to bind antibodies (IgGs) from different subclasses and species through the heavy chain in the Fc region. The two proteins differ in their binding properties for antibodies from different species and IgG subclasses: protein A is generally preferred for rabbit, pig, dog, and cat IgG, while protein G has better binding capacity for a broader range of mouse and human IgG subclasses (IgG1, IgG2, etc.).

The oriented and reversible immobilization of antibodies ensures high activity of the immobilized IgG without lengthy optimization of immobilization protocols or regeneration conditions. With a Ka of approximately 108 M−1 protein A/G–IgG complexes are sufficiently stable to yield surfaces with low drift that are suitable for a broad range of applications. Binding to fusion protein A/G is less pH-dependent than to either protein A or protein G alone. However, incubation with denaturing buffers or at slightly acidic or alkaline pH will break the interaction and results in quantitative regeneration of the chip surface, which can be repeated several hundred times.1

  1. J. Quinn, P. Patel, B. Fitzpatrick, B. Manning, P. Dillon, S. Daly, R. O’Kennedy, M. Alcocer, H. Lee, M. Morgan and K. Lang, Biosens. Bioelectron., 1999, 14, 587–595.

Protein A/G on linear polycarboxylate polymer (HC)

Recombinant fusion protein A/G (Mw around 50 kDa) is pre-immobilized in a 3-dimensional hydrogel consisting of a linear polycarboxylate with higher flexibility in its polymer chains than carboxymethyldextran. Recombinant protein A/G on these sensor chips consists of seven IgG-binding domains (EDABC–C2C3); the protein A portion (segments E, D, A, B, and C) is from Staphylococcus aureus, and the protein G portion (segments C2 and C3) is is a Streptococcal cell wall protein. Sensor surfaces with protein A/G are designed to bind antibodies (IgGs) from different subclasses and species through the heavy chain in the Fc region. The two proteins differ in their binding properties for antibodies from different species and IgG subclasses: protein A is generally preferred for rabbit, pig, dog, and cat IgG, while protein G has better binding capacity for a broader range of mouse and human IgG subclasses (IgG1, IgG2, etc.).

The oriented and reversible immobilization of antibodies ensures high activity of the immobilized IgG without lengthy optimization of immobilization protocols or regeneration conditions. With a Ka of approximately 108 M−1 protein A/G–IgG complexes are sufficiently stable to yield surfaces with low drift that are suitable for a broad range of applications. Binding to fusion protein A/G is less pH-dependent than to either protein A or protein G alone. However, incubation with denaturing buffers or at slightly acidic or alkaline pH will break the interaction and results in quantitative regeneration of the chip surface, which can be repeated several hundred times.1

  1. J. Quinn, P. Patel, B. Fitzpatrick, B. Manning, P. Dillon, S. Daly, R. O’Kennedy, M. Alcocer, H. Lee, M. Morgan and K. Lang, Biosens. Bioelectron., 1999, 14, 587–595.

Pectin (PC)

Pectins, also known as pectic polysaccharides, are structural heteropolysaccharides contained in the primary cell walls of terrestrial plants. Isolated pectin, which is rich in galacturonic acid, has a typical molecular weight of 60,000–100,000 Da, varying according to the extraction conditions and origin.

Pectin hydrogels are useful chip surfaces with low nonspecific background for the analysis of biomolecules expressed in plants.

Due to the abundant carboxyl groups, PC coatings are fully compatible with all immobilization chemistries available for CM-Dextran sensor chips.

Polyethylene glycol (PEG)

The PEG sensor surface coating is a hydrophilic, polar surface based on a dense brush of 4-kDa PEG. The low polymer–water interfacial energy value results in good resistance to protein adsorption and cell adhesion.

The PEG surface is hydroxyl terminated and uncharged. It can be activated by oxidation to aldehydes and subsequent Schiff base coupling/reductive amination, or by (bis)epoxy activation. As there will not be any electrostatic preconcentration, high ligand concentrations are necessary to reach a sufficient level of immobilization.

Due to the terminal functionalization of the PEG chains, this surface is useful for the preparation of ligand monolayers on a well-stabilized, semi-hydrated and passive spacer layer.

Poly-L-lysine (PLY)

Pure poly-L-lysine brush, 50–100 nm thick. High positive charge density. Sensor chips coated with PLY can be used for reverse electrostatic preconcentration and immobilization of amino reactive, negatively-charged ligands, polymers or biological structures, such as cells and tissue fragments. Note that the nonspecific background of this surface can be significant, as most biomolecules carry a negative charge at physiological pH.

Streptavidin derivatized carboxymethyldextran (SAD)

The streptavidin-modified carboxymethyl dextran matrix consists of covalently bound streptavidin in a biocompatible, hydrophilic polysaccharide. The 55-kDa streptavidin tetramer is composed of four identical polypeptide chains and is dissociated into monomers before ligand immobilization, which prevents baseline drift and leaching during the ligand regeneration steps.

Due to the mechanical rigidity of the polymer backbone, combined with branching and pronounced hydration, the movement of immobilized ligands in SAD polysaccharide hydrogels is limited. Therefore, SAD hydrogels are a good regime if the focus is on monovalent interactions. If immobilized ligands are expected to interact in a cooperative manner, the more flexible SAHC hydrogel should be used instead.

Streptavidin derivatized HC polycarboxylate (SAHC)

The streptavidin-modified HC matrix consists of covalently bound streptavidin in a bioinert, extremely hydrated, strictly linear polycarboxylate. The 55-kDa streptavidin tetramer is composed of four identical polypeptide chains and is dissociated into monomers before ligand immobilization, which prevents baseline drift and leaching during the ligand regeneration steps.

SAHC hydrogels exhibit a very low nonspecific background. The small molecular footprint of the HC polymer chains leaves sufficient space for unhindered diffusion of the analyte, even after immobilization of voluminous ligands. The polymer chains of the SAHC hydrogel are extremely flexible, giving immobilized ligands a high degree of freedom and the ability to interact in a cooperative manner with large analytes.

Streptavidin immobilized on 2D carboxymethyldextran (SAP)

The streptavidin-modified planar (two-dimensional, 2D) carboxymethyldextran matrix consists of covalently-bound streptavidin on a thin (<5 nm), biocompatible, hydrophilic polysaccharide cushion. The 55-kDa streptavidin tetramer is composed of identical polypeptide chains that are dissociated into monomers before ligand immobilization to prevent baseline drift and leaching during the ligand regeneration steps.

Because of the planar nature of the SAP structure, the immobilization capacity is significantly lower than that of 3D streptavidin coatings (LINK SAD, SAHC). However, the planar variant has significant advantages over the 3D hydrogels in the kinetic measurement of larger analytes such as antibodies. Because of the absence of flexible polymer strands, the immobilized ligands are spatially resolved on the surface and mainly monovalent. Furthermore, analytes on the planar SAP structure can diffuse unhindered to, and dissociate from, the surface without steric hindrance caused by 3D matrices. Moreover, analyte re-binding during the dissociation phase does not occur. All these factors mean that kinetic data obtained in measurements on the SAP surface are more reliable than comparable measurements made using 3D hydrogels.

Tetraethylene glycol (TEG)

Tetraethylene glycol (TEG) is a member of a homologous series of dihydroxy alcohols and is well known for its biocompatibility. The sensor chip coating consists of a TEG self-assembled monolayer (SAM), which exclusively presents hydroxyl groups on the surface. Sensor chips with TEG SAMs have the advantage that the interactions take place very close to the gold surface, and thus in the most sensitive part of the evanescent field, resulting in acceptable SPR signals with medium-weight molecules as analytes. The TEG surface is ideal for the development of biocompatible surfaces, and can be used as a reference because of its well-documented ability to suppress protein adsorption and plasma activation. Furthermore, TEG can be modified chemically to alter the properties of the surface1, 2. Use of modified TEG sensor chips is of interest in the pharmaceutical and cosmetics industries, as TEG is often used as an additive in their products.

For TEG-based sensor chips with some carboxyl groups on the surface, which allow easy and gentle immobilization via EDC/NHS chemistry, please select CMTEG sensor chips.

  1. Cha, T., Guo, A., Jun, Y., Pei, D., & Zhu, X. Y. (2004). Immobilization of oriented protein molecules on poly (ethylene glycol)‐coated Si (111). Proteomics, 4, 1965–1976.
  2. Piehler, J., Brecht, A., Valiokas, R., Liedberg, B., & Gauglitz, G. (2000). A high-density poly (ethylene glycol) polymer brush for immobilization on glass-type surfaces. Biosensors and Bioelectronics, 15, 473–481.

Disulfide modified hydrogels (THC, TD, TP)

THC, TD and TP sensor chips are coated with a disulfide derivatized bioinert hydrogel respectively 2D coating based either on carboxymethyldextran or the linear polycarboxylate HC. Covalent disulfide coupling requires reduction of surface and ligand disulfides and activation of the formed surface thiol groups with pyridyl disulfide before ligand injection. All THC, TD and TP coatings also contain carboxyl groups for electrostatic preconcentration of protein ligands.

UV-reactive sensor chip surfaces

The UV crosslinking surface makes immobilization very easy. Let your molecule of interest preconcentrate on the surface, then irradiate with near-UV light. A high immobilization yield and an immobilization chemistry that targets many functional groups such as hydroxyls and amines, as well as less reactive C–H and C–C bonds, allow the immobilization of carbohydrates and small molecules which are not accessible through traditional NHS/amine coupling. The wavelength and UV dose required for good coupling yields do not affect even sensitive ligands.

The UV chemistry is compatible with the Reichert 2SPR system equipped with a UV flow cell, and SPRi instruments using external spotters. Alternatively, it is possible to undock a sensor chip after ligand preconcentration, irradiate the surface with UV, and reinsert the chip to continue the experiment.

Zwitterionic surfaces (ZC, ZCX, ZCC)

This unique hydrogel is a derivative of the robust C polycarboxylate. It has been developed for covalent immobilization of negatively charged molecules such as DNA, carbohydrates and acidic proteins at physiological pH, employing a reverse electrostatic preconcentration effect. In contrast with the usually polyanionic surfaces for covalent immobilization, this surface exhibits a temporarily positive net charge during preconcentration and coupling of the negatively charged ligand. After coupling and quenching of the active NHS esters, the net charge becomes slightly negative again, thus stabilizing the surface against nonspecific interactions.

The zwitterionic hydrogel is available as an NHS-preactivated surface (ZCX) or a non-activated surface (ZC) to be activated in situ with standard EDC/NHS chemistry.

To determine preconcentration conditions(preconcentration scouting), we offer the positively charged ZCC version, which mimics the NHS activated surface. However, the ZCC surface is unreactive, so the ligand can be eluted multiple times until optimization of the coupling parameters has been completed.

Products | SPR Sensor chips | Hydrogels

Hydrogels

With a sufficiently sensitive detector, 2D surfaces are a good basis for many biomolecular interaction studies; however, numerous applications require a ligand density greater than one monolayer, either because the analyte is small (<2 kDa), or the optics are not sufficiently sensitive. In these cases, use of hydrogels can be advantageous.  A structurally flexible and strongly hydrated polymer brush is useful to shield the substrate and suppress non-specific interactions. Enhanced accessibility of the ligand’s binding sites and its increased mobility when immobilized through molecular spacers are further advantages of using hydrogels. Hydrogels can also help to solubilize hydrophobic ligands that would otherwise tend to form insoluble aggregates on the chip surface, and protect sensitive ligands against denaturation, especially in the dry state.

These advantages led to the development of surface-grafted, typically brush-structured, 3D hydrogels with thicknesses ranging from <10 nm to >1000 nm. These structures provide an increased density of attachment sites on the chip and, more importantly, use a higher volume fraction of the evanescent field for the specific interaction compared with 2D coatings. A welcome side effect of this approach is that the absolute influence of nonspecific factors such as bulk shifts decreases proportionally with increasing occupation of the evanescent field volume by the ligand–analyte pair.

In principle, surface-grafted hydrogels can be made from any water-soluble polymer; however, in practice, polycarboxylates, polyethers and polyols are preferred as they show significantly lower backgrounds than, for example, polyamines. In the early 1990s, the first commercial sensor chip coatings were derived from well-characterized, dextran-based solid phases for affinity chromatography. The most popular matrix material today is carboxymethylated dextran. Other carboxylated polysaccharides such as alginate, pectin, carboxymethylcellulose and hyaluronic acid can be used as well, but are less common. Because these polymers are of natural origin, their structures are often irregular; i.e., they can be more or less branched, or form hyperstructures such as helices or suprafibers. Furthermore, especially in the hydrated state, carbohydrates are relatively bulky molecules, occupying a considerable volume of the evanescent field that is then not available for the immobilization of ligand, and hindering the free diffusion of analyte.

For these and other reasons, efforts have been made to substitute polysaccharides with better defined synthetic polycarboxylates or zwitterionic polymers with lower background and smaller molecular footprints. In addition to reducing steric issues, coatings containing no hydroxyl groups have the advantage that they cannot form ester crosslinks upon EDC/NHS activation, a common side reaction occurring with carboxymethylated carbohydrates.

Hydrogel surfaces

3D immobilization matrices allow multilayer ligand immobilization which leads to significant signal amplification. They are employed for qualitative screening, concentration determinations and detection and kinetic analysis of low molecular weight compounds. As a rule of thumb, the smaller the analyte is, the thicker and denser the hydrogel structure should be.

The new hydrogel coatings of the HC family are currently available in four different thickness / porosity combinations: HC30M, HC200M, HC1000M and HC1500M. The HC type is based on a linear, synthetic polycarboxylate. Compared to the branched dextrans, it is better defined, shows a higher signal to noise ratio, has improved diffusion properties (better curve fits) and lower nonspecific interactions. Even more inert is the new HLC version which is available in the same thickness density combinations.

Hydrogel surfaces
Product descriptionProduct code
Alginate hydrogel.AL
Approx. 50 nm
Azide derivatized carboxymethyldextran hydrogel.AZD x
For click coupling. 50L, 200M
Azide derivatized linear polycarboxylate hydrogel.AZHC x
For click coupling. 30M, 200M, 1500M
Biotin derivatized carboxymethyldextran hydrogel.BD x
200M
Biotin derivatized linear polycarboxylate hydrogelBHC x
200M
Linear polycarboxylate hydrogel, high charge density.C x
30M, 80M, 150D
Carboxymethyldextran hydrogel.CMD x
50L, 50M, 200L, 200M, 500L, 500M, 700L, 700M
Carboxymethylpolyethyleneglycol.CMPEG
4 kDa
Dextran hydrogel.D x
200M
DBCO derivatized carboxymethyldextran hydrogel, medium density.DCD x
For Click Coupling. 50L, 200M
Polycarboxylate hydrogel, medium charge density.HC x
Protein immobilization, standard. 30M, 200M, 1000M, 1500M
Heparin.HEP
Approx. 50 nm hydrogel.
Polycarboxylate hydrogel, low charge density.HLC x
Extremely low level of non-specific interactions. 30M, 200M, 1500M
Chloroalkane derivatized carboxymethyldextran hyrogel.HOD x
For coupling of HaloTag fusion proteins. 200M
Chloroalkane derivatized linear polycarboxylate hydrogel, medium densityHOHC x
For coupling of HaloTag fusion proteins. 200M
Carboxymethyldextran hydrogel, partially alkyl derivatized.LD
For immobilization of liposomes and vesicles.
Neutravidin derivatized linear polycarboxylate hydrogel, low charge density.NAHLC x
30M, 200M, 1500M
NTA derivatized carboxymethyldextran hydrogel.NID x
50L, 200M
NTA derivatized linear polycarboxylate hydrogel, medium charge density.NIHC x
30M, 200M, 1000M, 1500M
Protein A derivatized carboxymethyldextran hydrogel.PAD x
50L, 200L
Protein A derivatized linear polycarboxylate hydrogel.PAHC x
30M, 200M
Protein A/G derivatized carboxymethyldextran hydrogel.PAGD x
50L, 200L
Protein A/G derivatized linear polycarboxylate hydrogel.PAGHC x
30M, 200M
Poly-L-lysine.PLY
Streptavidin derivatized carboxymethyldextran hydrogel.SAD x
50L, 200L, 200M, 700L
Streptavidin derivatized linear polycarboxylate hydrogel.SAHC x
30M, 200M, 1500M
Spycatcher derivatized carboxymethyldextran hydrogel, medium densitySCBS SPYD x
For immobilization of SpyTag fusion proteins. 200M
Spycatcher derivatized linear polycarboxylate hydrogel, medium densitySCBS SPYHC x
For immobilization of SpyTag fusion proteins. 200M
Strep-Tactin XT derivatized carboxymethyldextran hydrogel.SCBS STD x
For reversible immobilization of Strep-tagged ligands. 200L
Strep-Tactin XT derivatized linear polycarboxylate hydrogel.SCBS STHC x
For reversible immobilization of Strep-tagged ligands. 200M
UV photocrosslinker derivatized carboxymethyldextran hydrogel, λ: 365 nm (UV-C)UVD x
200M
UV photocrosslinker derivatized polycarboxylate hydrogel, λ: 365 nm (UV-C)UVHC x
200M
Zwitterionic polycarboxylate hydrogel, cationic after NHS activation.ZC x
Immobilization of anionic ligands. 30M, 80M, 150D
Zwitterionic polycarboxylate hydrogel, cationic, non-activated.ZCC x
Preconcentration scouting of anionic ligands. 150D

The above mentioned SPR substrates are mainly intended for biomolecular interaction analysis, but may also be used as intermediate platforms for further modifications. The 2D and 3D coatings are covalently coupled to a protective interlayer and resist non-oxidative aqueous solutions from pH 1 - 13 as well as most organic solvents.

Alternative formats and custom coatings available upon request.