Biacore Sensor Chips: Types, Applications, and Selection Guide

The Biacore sensor chip is the core component of the Biacore system to achieve surface plasmon resonance technology, which is mainly used to fix the target molecule (ligand) and detect its interaction with the analyte. The chip acts as a reaction platform, providing a functionalized surface that allows molecules to be stably and controllably bound to the chip surface. During the experiment, the Biacore instrument monitors the molecular binding and dissociation process in real-time as the analyte solution flows over the chip surface, and then calculates the kinetic parameters (such as the binding rate constant, dissociation rate constant, and affinity constant). The chip's performance directly affects the sensitivity, stability, and repeatability of the experiment, which is the key to achieving high-quality data acquisition.

Overview of Biacore Sensor Chips

Biacore sensor chips offer a variety of fixed options to meet different molecular interaction analysis needs. CM5 chip is equipped with a carboxymethyl glucan matrix, which is suitable for fixing proteins, antibodies, small molecules, and nucleic acids through amine coupling. It is suitable for a wide range of biomolecular analysis. The NTA chip achieves efficient capture of His label proteins via the Ni2+ -NTA complex, suitable for the study of metal-chelated proteins and protein complexes. The SA chip uses streptavidin on the surface to achieve stable binding of biotinized molecules and supports reversible fixation. Protein A chip provides the specific trapping function of the Fc region of the antibody realizes the directed fixation of the antibody, and improves the accuracy of binding analysis. The CAP chip can be used to rapidly and reversibly capture nucleic acids, supporting DNA/RNA hybridization experiments. You can improve the efficiency of your experiments by choosing a sensible sensor chip, or you can get high-quality interaction data directly from our services.

How the sensor chip works

The Biacore sensor chip is based on the principle of surface plasmon resonance, which monitors molecular interactions in real-time without labeling molecules. The structure of the chip consists of the following parts:

Glass substrate: provides physical support and mechanical stability.

Metal coating (usually gold film): As a core part of SPR excitation, supporting the generation of surface plasma waves.

The functional surface layer (e.g. Carboxymethylated dextran matrix): provides rich functional groups (e.g. -COOH) for fixing ligands.

When light hits the metal layer on the back of the chip at an Angle, it excites plasma waves on the surface. The local refractive index changes when the molecule is bound on the chip surface, resulting in the SPR resonance Angle shift. The Biacore instrument senses these shifts by monitoring changes in the intensity of the reflected light and converting it into a real-time sensing signal (expressed in response units RU). The change in RU value is related to the mass of the binding molecule, so it can be used to calculate the binding amount and kinetic parameters.

Common chip types include CM5, NTA, SA, Protein A, and CAP chips, which are designed according to different fixation modes and application requirements. CM5 chip uses a carboxymethylated glucan matrix, which is suitable for a variety of fixation methods such as amine group coupling, and is widely used in the fixation of proteins, antibodies, small molecules, and nucleic acids. The NTA chip uses the Ni2+ -NTA complex to capture proteins with His tags; The SA chip is designed to bind biotinized molecules to achieve stable and reversible fixation. Protein A chip achieves directional fixation of antibody by binding specifically to the Fc region of antibody, which is suitable for antibody analysis. The CAP chip provides the ability to reversibly capture nucleic acid molecules and is suitable for DNA/RNA analysis. Each of these chips has its own characteristics, and researchers can choose the right type according to the experimental needs to obtain reliable and high-quality interaction data.

CM5

Main features

The CM5 sensor chip is one of the most widely used chips in the Biacore system and is suitable for a variety of molecular fixation methods, especially when covalent fixation ligands are required. Its surface is covered by a matrix of Carboxymethylated Dextran on a metal coating, providing rich carboxyl functional groups that can be used in a variety of coupling chemistry, such as amine coupling, sulfhydryl coupling, and hydroxyl coupling. The chip is designed to fix proteins, antibodies, nucleic acids, small molecules, sugars, etc., and is widely used in molecular dynamics analysis, affinity determination, and concentration analysis.

Key features of CM5 chip

Strong versatility: suitable for a variety of coupling methods, supporting the fixation of proteins, nucleic acids, small molecules, and polysaccharides.

High fixation capacity: Carboxymethylated dextran matrix can achieve high-density fixation and improve experimental sensitivity.

Wide application range: can be used for affinity determination, kinetic analysis, concentration determination, and competition experiments.

Good reproducibility: It can be reused through appropriate regeneration conditions to extend the service life of the chip.

Figure 1: CM5 chip product diagram. (fig from Cytiva)

NTA

Main features

The NTA sensor chip is a specialized chip in the Biacore system for fixing proteins with multi-histamine tags (His tags). The surface is covered with Nitrilotriacetic acid (NTA) groups, which form an active surface with the coordination of transition metal ions (usually Ni²⁺ his), thereby achieving reversible fixation of the protein through specific coordination between Ni² + -His labels. This fixation method does not require a chemical coupling, is simple to operate, gentle to fix, and easy to regenerate, and is ideal for analysis of protein-molecular interactions, protein screening, and membrane protein studies.

Key features of NTA chip

Reversible fixation, simple operation: His tag protein is quickly captured by Ni2+ activation, suitable for experiments requiring frequent ligand changes.

Mild fixation protects protein activity: non-covalent binding avoids the potential effects of chemical modifications on protein function.

Suitable for a variety of analytical applications: It can be used for the study of protein-protein, protein-small molecule, and protein-nucleic acid interactions.

High repetition rate: Short fixation and regeneration cycle, easy to carry out multiple rounds of experiments.

SA

Main features

The SA sensor chip is a sensor chip in the Biacore system specially used for fixing biotin-labeled molecules. Its surface is covered with a highly purified layer of Streptavidin (SA), which is able to form high affinity and extremely stable non-covalent binding to Biotin molecules. Due to the very low-affinity constant between biotin-streptavidin (about 10⁻¹⁵ M), this binding method is extremely robust and suitable for long-term analyses or experiments requiring highly stable ligand fixation.

Key features of SA chip

Ultra-high affinity binding: Biotin-streptavidin binding is extremely stable, and suitable for long-term, multi-round experiments.

Mild fixation retains molecular activity: Biotin labeling is easy to operate, and suitable for sensitive protein or nucleic acid fixation.

Wide range of applications: biotin-labeled proteins, nucleic acids, peptides, and small molecules can be fixed.

Low non-specific binding: The SA layer is stable and uniform, reducing background signal interference.

Figure 2: SA chip product diagram. (fig from Cytiva)

Protein A

Main features

The Protein A sensor chip is a chip designed for immobilizing immunoglobulin G (IgG) class antibodies in the Biacore system. Protein A is covalently coupled on the surface of the chip, which can specifically bind the Fc region of the IgG molecule to achieve the directed fixation of the antibody. This fixation method can preserve the availability of antigen-binding sites of antibodies and improve the sensitivity and repeatability of binding analysis. Because Protein A has a good affinity with IgG from a variety of species, the chip is suitable for applications such as antibody screening, antibody-antigen interaction studies, and antibody quality control.

Key features of Protein A chip

Directed fixation of IgG antibodies: Protein A binds the Fc region, keeping the Fab region available for antigen binding.

High affinity binding, easy operation: no antibody modification, directly fixed natural IgG molecules.

Wide range of applications: suitable for a variety of species of IgG, especially human and rabbit IgG.

High repeatability and regeneration: Multiple rounds of combined analysis can be performed under mild regeneration conditions.

CAP

Main features

CAP sensor chip is a chip designed for nucleic acid molecular fixation and analysis in the Biacore system. Its surface is prefixed with a specific Capture Reagent, which can be efficiently combined with biotin-labeled Capture oligonucleotides (Capture Probe), so as to achieve indirect fixation of target nucleic acids. This two-step capture mechanism (the CAP molecule binds to a biotinized capture probe, which is then paired with a complementary nucleic acid) enables reversible fixation of nucleic acid molecules and provides high stability and reproducibility for the study of DNA, RNA, and their interactions with proteins or small molecules.

Key features of CAP chip

Designed for nucleic acid fixation: Highly efficient capture of biotin-labeled oligonucleotides for DNA/RNA analysis.

Flexible reversible fixation: The capture probe can be removed and replaced to support multiple rounds of experiments.

Fixed mild reactivity: do not directly modify the target molecule, maintain the natural structure and function of nucleic acid.

High specificity and low background: The capture system design reduces non-specific adsorption, and the data is stable and reliable.

Choosing the Right Chip

Choosing the right Biacore sensor chip is critical to ensure the success of the SPR experiment. Different chips have their own advantages according to the fixation mechanism, target molecule type, experimental needs, and regeneration requirements.

Select according to the fixed mode

Covalently fixed (e.g. CM5 chip)

Application scenario: long-term experiment, multi-round combination analysis, high stability requirements.

Advantages: Ligands are permanently fixed, and suitable for a wide range of molecular fixation (proteins, small molecules, peptides, etc.).

Limitations: Fixed molecules cannot be replaced after regeneration.

Non-covalent fixation (e.g. SA, NTA, Protein A, CAP chips)

Application scenarios: Experiments requiring flexible ligand replacement or gentle fixation.

Advantages: Easy to operate, some chips support the regeneration and replacement of captured molecules.

Limitations: Fixation stability may be lower than covalent fixation

Select according to the purpose of the experiment

Analysis of protein-protein interaction

Recommended chips: CM5 (Long term experiment), NTA (rapidly reversible fixation), Protein A (when the antibody is ligand). CM5 provides stable fixing; NTA is suitable for rapid protein change; Protein A supports antibody-directed fixation.

Nucleic acid binding study

Recommended chips: CAP (for nucleic acid capture), SA (fixed biotin-labeled nucleic acid). CAP supports probe replacement and is suitable for multi-target analysis. SA is suitable for experiments that require extremely high fixed stability.

Antibody screening and binding kinetics assay

Recommended chips: Protein A (direct fixation IgG antibody), CM5 (long-term antibody fixation assay). Protein A is simple to operate, and targeted antibody fixation improves binding activity.

Small molecule drug screening

Recommended chips: CM5 (fixed protein target), SA (fixed biotin-labeled protein), CAP (nucleic acid target screening). CM5 provides a stable platform for high-throughput drug screening.

Amine Coupling and Immobilization

The functionalized surface of the chip supports a variety of immobilization methods, the most common of which is amine coupling:

Surface activation: activation of carboxyl groups by EDC/NHS into intermediates that can react with amino groups.

Ligand injection: Ligands containing amino groups flow across the surface to form stable amide bonds with the activation site.

Blocking procedure: Use ethanolamine to block the unreacted site and reduce non-specific binding.

This fixation method makes the ligands firmly bound to the surface of the chip and can be used for multi-wheel binding and regeneration experiments. Other fixation methods, such as sulfhydryl coupling and affinity capture, offer different fixation options to suit different types of molecules and experimental needs.

Maintenance and Regeneration of Chips

In order to ensure the long-term effective use of Biacore sensor chips and the reliability of experimental data, a reasonable chip maintenance and regeneration strategy is essential. Proper regeneration methods can remove the binding without damaging the fixed ligand, while proper maintenance measures can significantly extend the service life of the chip and reduce experimental errors. CM5 chip can support multiple cycles of regeneration and is easy to operate, which is suitable for long-term experiments. Although NTA and CAP chips support reversible fixation and probe replacement, attention should be paid to metal ion reloading or probe integrity during regeneration. The Protein A chip can be gently regenerated and is suitable for antibody-related experiments, while the SA chip needs to be fully considered in the fixed design stage due to the irreversibility of biotin binding.

Maintenance

The chip surface should be cleaned with deionized water and running buffer before and after the experiment to prevent residue accumulation. The chip should be kept wet when not in use and can be left in the system for short-term circulation storage or long-term storage at 4°C and sealed storage. Avoiding the chip surface drying and repeated freeze-thaw is the key to preventing damage to the fixed layer. In addition, to prevent contamination, a regular cleaning with 50 mM NaOH or low-concentration SDS can remove organic matter, lipid residues, or salt deposits.

Regeneration

Effective maintenance and regeneration are key steps to extend the service life of the Biacore sensor chip and ensure the reliability of the experimental data. The main purpose of chip regeneration is to remove the binding and restore the binding ability of the sensing surface while keeping the fixed ligand activity undamaged. The choice of regenerated solution is based on the type of binding molecule and the fixation mechanism to ensure that the binding can be effectively removed without damaging the stability of the chip surface. For example, protein-protein binding is usually regenerated using Glycine-HCl (pH 1.5-3.0), while small-molecular-protein binding may require 50 mM NaOH or a low-concentration SDS solution to remove tightly bound small molecules. For NTA chips, regeneration of His tag protein binding can be done by removing the binding protein using 350 mM EDTA and re-injecting NiCl₂ to restore capture. Because of the high stability of biotin binding, SA chips are usually not suitable for regeneration methods.

During the regeneration process, the concentration and action time of the regenerated solution should be adjusted according to the binding strength and ligand tolerance of the target molecule. The usual procedure involves injecting the regenerated solution (30-60 seconds) at the end of the binding experiment and then quickly flushing with the running buffer to prevent prolonged exposure of the ligand to the regenerated solution. The baseline after regeneration should be close to the initial level, if it fails to recover, it is necessary to optimize the regeneration conditions or change the type of regeneration fluid. Small-scale tests are recommended to verify repeatability and ligand stability before multiple rounds of regeneration experiments.

Table 1 Selection principle and explanation of regenerated liquid