Biacore SPR Technology and System Components

Introduction to Biacore Technology

Biacore SPR technology allows researchers to measure the binding interactions between biomolecules—proteins, nucleic acids, peptides, small molecules, and even cells—without the need for labels or complex sample preparation. SPR works by detecting changes in the refractive index at the surface of a sensor chip when biomolecules interact with an immobilized ligand.

Since its inception in the late 1980s, Biacore has set the standard for SPR technology, providing high sensitivity and the ability to capture real-time kinetic data. Whether it's determining the binding affinity of a drug candidate or evaluating the interactions between antibodies and antigens, Biacore SPR systems offer critical insights into the dynamics of molecular interactions.

Main Components of the Biacore System

The Biacore system is composed of several interrelated components that work together to provide high-quality data. These components are essential for ensuring accuracy, sensitivity, and reproducibility during molecular interaction analysis.

SPR System

At the heart of the Biacore system is the SPR sensor, which uses surface plasmon resonance to detect changes in the refractive index of a sensor chip when biomolecules bind. The SPR system consists of several key elements, including an optical system that directs polarized light onto a metal surface (usually gold) at a precise angle, generating surface plasmons.

When a molecule binds to the sensor chip, it alters the local refractive index, which shifts the angle of plasmon resonance. This change in resonance is measured in real-time and plotted as a sensorgram, providing a visual representation of the interaction kinetics.

The system includes:

• Light Source: Emitting near-infrared light, which interacts with the sensor chip to generate SPR signals.
• Sensor Chip: A thin glass plate coated with a gold layer, which provides the surface for immobilizing one of the interaction partners (ligand).
• Automated Liquid Handling System: This system ensures that the analyte (the second binding partner) is directed over the sensor surface in a continuous flow, facilitating the real-time monitoring of interactions.
• Diode Array Position-Sensitive Detector: It measures the shift in the plasmon resonance angle, quantifying the change in refractive index as analytes bind to the immobilized ligand.

Key Functions:

• Measures binding events in real-time, offering unmatched sensitivity.
• Capable of detecting even low-affinity interactions.
• Provides direct, quantitative information on molecular interactions, such as affinity and kinetics (on-rate and off-rate).

Detection Unit

The detection system in Biacore monitors molecular interactions in real time by detecting changes in the refractive index at the surface of the sensor chip. These changes occur when molecules bind to the immobilized ligand, and they are directly proportional to the molecular mass and concentration of the analyte.

The system produces a sensorgram, which is a plot of the resonance units (RU) over time, representing the interaction dynamics:

• Association Phase: This occurs when the analyte binds to the immobilized ligand, causing an increase in the SPR signal.
• Dissociation Phase: After the analyte is removed, the complex dissociates, and the SPR signal decreases. If the dissociation rate is slow, the complex may need to be dissociated by a specific regeneration buffer.
• Regeneration: The surface is regenerated to remove bound molecules, allowing for repeated cycles of analysis. This is particularly important when performing kinetic studies to measure the rates of association and dissociation.

The SPR signal is captured by a diode array position-sensitive detector, which accurately measures small shifts in the angle of the surface plasmon resonance. This high sensitivity allows the system to detect even the most subtle molecular interactions, making it ideal for studying low-affinity interactions or small molecules.

Key Functions:

• Captures SPR signals with high sensitivity.
• Measures real-time binding and dissociation events with high precision.
• Ensures accurate data interpretation, even in complex assays.

Flow System

The microfluidic flow system delivers the sample solutions (analytes) over the sensor chip in a highly controlled manner. It ensures that the analyte flows over the immobilized ligand on the sensor chip at a constant rate, creating reproducible experimental conditions. The flow system is essential for maintaining steady-state conditions during the assay, as well as ensuring that the sample is introduced in a manner that avoids introducing error.

Key Functions:

• Delivers analyte samples over the sensor chip with high precision.
• Regulates the flow rate and ensures consistent buffer delivery, critical for reproducible results.
• Enables automated sample handling for high-throughput applications.

Sensor Chips

The sensor chip is where the molecular interactions take place. A variety of sensor chips are available for Biacore systems, each designed for different types of analyses and interactions. Sensor chips are typically coated with a thin layer of gold, which supports the SPR phenomenon. Each chip is functionalized to enable the immobilization of one of the interacting molecules—often a protein or antibody.

Types of Sensor Chips:

• CM5 Chip: The most commonly used chip for proteins and other biomolecules. It provides a versatile surface chemistry for the immobilization of a wide range of molecules.
• SA Chip: Coated with streptavidin for the immobilization of biotinylated molecules. Ideal for antibody-antigen binding studies.
• NTA Chip: Designed specifically for the immobilization of His-tagged proteins. Useful for protein-protein interaction studies.
• Protein A/G Chips: These chips are specifically used for the immobilization of antibodies, making them ideal for immunological assays.

Key Functions:

• Supports immobilization of interacting molecules for SPR analysis.
• Available in various configurations to accommodate different types of biomolecular interactions.
• Allows high precision in binding assays, minimizing nonspecific interactions.

Immobilization

The immobilization of the ligand onto the sensor chip surface is a crucial step in Biacore experiments. There are various methods for coupling ligands to the chip surface, depending on the nature of the ligand and the experimental requirements:

• Covalent Coupling (Amine Coupling): This is the most common approach and involves the use of primary amines (e.g., from lysine residues) on the ligand, which react with the activated surface of the chip. This method provides a stable, non-reversible attachment of the ligand.
• Non-Covalent Coupling (Reversible): This method relies on high-affinity binding interactions, such as the use of antibodies as capture molecules. In this case, the antibody binds to the ligand and can be dissociated later for regeneration of the surface.
• Surface Thiol Coupling: This method utilizes thiol groups on either the sensor surface or the ligand. Thiols on the surface can interact with reactive disulfide groups introduced to the ligand, enabling stable coupling for certain types of biomolecules.

For example, in an anti-GST antibody immobilization procedure, the antibody is covalently coupled to the sensor surface via amine coupling, and the amount of immobilized antibody is quantified by measuring the change in Resonance Units (RU) before and after surface deactivation.

Data Analysis Software

Biacore systems come with advanced data analysis software that interprets the sensorgram data captured during the experiment. The software provides powerful tools for analyzing the kinetic and affinity properties of molecular interactions. It uses mathematical models to derive key parameters such as the association rate constant (k_on), dissociation rate constant (k_off), and equilibrium dissociation constant (K_D).

Key Functions:

• Analyzes sensorgrams to calculate kinetic and affinity parameters.
• Offers advanced modeling and fitting options (e.g., 1:1 binding model, bivalent analyte model).
• Provides real-time data analysis with error detection and correction tools.
• Generates detailed reports for publication or further research.

Detection system of SPR based Biacore Flexchip (left) and a sensorgram of a typical antibody-peptide interaction (right) (Köhler et al., 2012).

Comparison of Different Biacore Models

Biacore offers a range of systems to suit different research needs, from high-throughput drug discovery to academic research. While all Biacore systems utilize SPR technology, they differ in their capabilities, throughput, and sensitivity.

Biacore T200

The Biacore T200 is one of the most widely used models, offering a balance of sensitivity, versatility, and ease of use. It features a multi-channel system that allows for simultaneous analysis of multiple samples, making it suitable for both kinetic and affinity measurements.

Key Features:

• Multi-channel system (up to 4 channels).
• Ideal for studying both small molecules and large biomolecular interactions.
• High sensitivity for detecting low-affinity interactions.
• Suitable for applications like drug discovery, antibody characterization, and protein-protein interactions.

Biacore 8K

The Biacore 8K is designed for ultra-high sensitivity and throughput. It features an eight-channel system that can simultaneously analyze multiple interactions, making it particularly useful in high-throughput screening (HTS) and complex biomolecular interaction studies.

Key Features:

• Eight-channel capability for parallel analysis.
• High sensitivity for detecting weak interactions.
• Fast analysis for high-throughput workflows.
• Ideal for large-scale drug screening and complex biomolecular studies.

Biacore X100

The Biacore X100 is a compact, benchtop system that is well-suited for researchers who need a reliable and cost-effective SPR system. It provides essential SPR functionality with high-quality data output, making it an excellent choice for routine assays.

Key Features:

• Small, user-friendly platform for basic SPR assays.
• Suitable for small to medium-scale studies.
• Affordable, making it ideal for academic and early-stage research labs.

Biacore 4000

The Biacore 4000 is designed for more complex applications that require high throughput and sensitivity. It can handle intricate kinetic and affinity measurements, making it suitable for large-scale drug development and detailed biomolecular research.

Key Features:

• High throughput for complex, multi-sample analyses.
• Suitable for high-affinity and low-affinity studies.
• Ideal for advanced drug discovery applications and epitope mapping.

Table 1. Comparison of Biacore Models

Reference

1. Köhler, Katja & Seitz, Harald. (2012). Validation Processes of Protein Biomarkers in Serum—A Cross Platform Comparison. Sensors (Basel, Switzerland). 12. 12710-28. 10.3390/s120912710.