Surface Plasmon Resonance (SPR) vs Biolayer Interferometry (BLI) Which is Better

In the field of biomolecular interaction research, surface plasmon resonance (SPR) and biosphere interferometry (BLI) are two kinds of marker-free detection techniques that have attracted much attention. With their unique principles and advantages, they play an important role in drug development, protein interaction research, antibody screening, and other fields.

Based on the surface plasmon resonance phenomenon, SPR technology monitors the intermolecular bonding and dissociation process in real time by detecting the change in the refractive index of the sensor surface. Its high sensitivity and precise kinetic data make it an ideal tool for drug screening, protein interaction studies, and small-molecule compound analysis. However, SPR equipment is expensive, complex to operate, and requires specialized technical support.

In contrast, the BLI technique is based on the principle of light interference and analyzes molecular interactions by measuring changes in the thickness of biomolecular layers. BLI is easy to operate and fast to test, which is suitable for high-throughput screening and rapid detection. However, its sensitivity and kinetic data accuracy are relatively low, which is more suitable for preliminary combination experiments or large-scale screening scenarios.

This article will compare the principles, advantages, and disadvantages of SPR and BLI in detail, and help you choose the most appropriate technology according to the research objectives, so as to improve the experimental efficiency and data reliability.

Comparison of SPR and BLI Technology Principles

What is SPR?

Surface plasmon resonance is a highly sensitive labeling-free biomolecular interaction analysis technique. The principle is based on the fact that when polarized light illuminates the surface of a metal film (such as a gold film), it will excite the surface plasmon resonance at a specific angle, resulting in a significant decrease in the intensity of the reflected light. When the molecule binds to the sensor surface, it causes a change in the local refractive index, which changes the resonance Angle. By monitoring changes in this Angle in real time, SPR can accurately measure intermolecular binding and dissociation processes, provide kinetic data (such as binding rate, dissociation rate, and affinity), and is widely used in protein-protein, protein-small molecule, antibody-antigen interaction studies. SPR preserves the natural state of the molecule without labeling the sample, making it an ideal tool for high-precision biomolecular research. For more details, see A Comprehensive Guide to Biacore Instruments Features, Specifications, and Applications.

What is BLI?

Biosphere interferometry is a label-free biomolecular interaction analysis technique based on the principle of light interference. The core principle is to analyze the binding between molecules by measuring the thickness change of the biomolecular layer at the tip of the sensor. When light passes through a sensor coated with a biomolecular layer, it forms an interference spectrum; When the target molecule binds to the biomolecules on the sensor surface, the thickness of the molecular layer changes, resulting in a shift in the interference spectrum. By detecting this shift, BLI can indirectly measure the degree to which molecules are bound.

The advantage of BLI is that it is easy to operate, fast, suitable for high-throughput screening, and without complex fluid systems, the sample can be directly immersed in the sensor to complete the detection. However, its sensitivity and kinetic data accuracy are relatively low, which is more suitable for preliminary combination experiments or large-scale screening scenarios.

Difference Between SPR and BLI

SPR and BLI are two common labeling-free biomolecular interaction analysis techniques, and their principles and application scenarios have their own characteristics.

The core of SPR technology lies in the surface plasmon resonance phenomenon. When polarized light hits the surface of a metal film (usually gold film), it will excite the surface plasmon resonance at a specific Angle, resulting in a significant reduction in the intensity of the reflected light. When the molecule binds to the sensor surface, it causes a change in the local refractive index, resulting in a shift in the resonance Angle. By monitoring the change of the resonance Angle in real-time, the process of molecular binding and decoupling can be accurately measured. The advantage of SPR is its high sensitivity and real-time monitoring capability, which enables the detection of extremely low concentrations of molecular interactions and provides detailed kinetic data such as binding rate, dissociation rate, and affinity. In addition, SPR does not require fluorescence or Radiolab elation of the sample and maintains the natural state of the molecule, which is suitable for the study of protein-protein, protein-small molecule, antibody-antigen, and other interactions.

In contrast, the BLI technique is based on the principle of light interference and analyzes molecular interactions by measuring changes in the thickness of biomolecular layers. The tip of the sensor is coated with a biomolecular layer that forms an interference spectrum when light passes through the sensor. When the molecules bind to the sensor surface, the thickness of the biomolecular layer changes, resulting in a shift in the interference spectrum. By detecting this shift, the degree of molecular binding can be measured indirectly. The advantage of BLI is its simple operation and rapid detection capability, which is suitable for high-throughput screening. Since there is no need for a complex fluid system, the sample is immersed directly into the sensor, reducing the number of operating steps. However, the sensitivity of BLI is low, the accuracy of kinetic data is not as good as that of SPR, and the detection ability of low-concentration samples is limited, so BLI is more suitable for preliminary combination experiments or large-scale screening.

The following table summarizes their key characteristics.

Table 1. Comparison of technical characteristics of SPR and BLI

Advantages and Disadvantages of SPR and BLI

SPR Technology

Advantages

• High sensitivity: The SPR is able to detect very low concentrations of molecular interactions and is suitable for studying low-abundance samples.
• Real-time monitoring: The SPR provides real-time kinetic data, including binding rates, dissociation rates, and affinity, to provide insight into the mechanisms of molecular interactions.
• Label free-detection: there is no need to fluorescent or radiolabelling the sample, maintaining the natural state of the molecule and reducing experimental interference.
• Wide application: Suitable for a variety of molecular types, including proteins, small molecules, nucleic acids, cells, etc., covering drug screening, protein interaction research, antibody characterization, and other fields.
• High-precision data: The SPR provides high-quality combined kinetic and thermodynamic data for research requiring high-precision results.

Shortcomings

• High equipment cost: SPR instruments are expensive and the initial investment is large.
• Complex operation: Professional technical personnel are required for operation and data analysis, and the learning curve is steep.
• Higher sample consumption: SPR experiments may require a larger sample size than BLI.
• Fluid system limitations: The SPR relies on fluid systems and may have issues such as clogging or bubble interference.

BLI Technology

Advantages

• Simple operation: BLI instrument has a compact design and easy experimental operation, suitable for quick use.
• High-throughput: Support multi-channel parallel detection, suitable for large-scale screening and fast experiments.
• No fluid system required: The sample is immersed directly into the sensor, reducing complex handling and potential fluid system problems.
• Rapid detection: BLI experiments are usually short in time and are suitable for scenarios that require fast results.
• Lower cost: Compared to the SPR, the BLI instrument is more affordable and suitable for research teams with limited budgets.

Shortcomings

• Low sensitivity: BLI has a limited ability to detect low-concentration samples and is not suitable for studies requiring high sensitivity.
• Limited accuracy of dynamic data: The accuracy of dynamic data (such as binding rate and decoupling rate) provided by BLI is not as good as that of SPR, which may not meet the needs of high-precision research.
• Narrow scope of application: BLI is more suitable for preliminary binding experiments or high-throughput screening, and has limited ability to study complex molecular interactions.
• Low sample compatibility: Some complex samples (such as cells or crude extracts) may not be suitable for BLI testing.

Application Scenario and Selection of SPR and BLI

Examples of SPR Application Scenarios

In anti-cancer drug development, researchers use SPR to monitor the binding dynamics of small molecule drugs to target proteins (such as kinases) in real-time, accurately determining binding rates, dissociation rates, and affinity to optimize drug design. For example, Mulyanti B, et al. used fiber-based SPR and LSPR to detect kidney disease early by detecting analytes associated with kidney disease, including urea, creatinine, glucose, uric acid, and dopamine.

In medicinal chemistry research, SPR is used to screen libraries of small molecule compounds and evaluate their binding ability to target proteins. In a classic example, Wang Q et al., based on previous knowledge and experience in treating similar diseases, researchers have come up with hundreds of possible drug candidates in the shortest possible time. Based on surface plasmonic resonance technology, this paper summarizes the application of SPR technology in COVID-19 research from four aspects: the way SARS-CoV-2 invades host cells, antibody candidates for COVID-19 treatment, small molecule drug reuse, and COVID-19 vaccine.

SPR technology is becoming a powerful tool for studying the interaction between drugs and targets due to its high efficiency, automation, marker-free, and high-resolution data. SPR technology can not only obtain the affinity data between drugs and targets but also clarify the binding sites and mechanisms of drugs.

Examples of BLI Application Scenarios

Bioluminescence imaging is a newly developed visual method in scientific research that is particularly useful for exploring a large number of biological processes in the body. Bioluminescence produced by the reaction between luciferase and luciferin can penetrate the skin and tissues, enabling the reading of physiological processes. So far, several bioluminescent systems composed of natural luciferin and its corresponding enzymes have been successfully applied in BLI, such as coelentin-dependent systems and firefly luciferin-luciferase systems.

A classic example is the wealth of information it can provide for the diagnosis, treatment, and prognosis of cancer. Several bioluminescent probes that can be recognized and activated by tumor-specific enzymes have been successfully used to treat and detect cancer, such as Z-DevD-NH2-LH2, Ac-RYKR/Ac-RVRR, and luciferase IPA. In recent years, a number of novel bioluminescent probes for the detection of tumor-specific enzymes have been reported. For example, Li et al. Three bioluminescent probes, L-PHE-LEU-NH2-LH2, L-LEU-NH2-LH2, and L-AlA-NH2-LH2, were designed. By coupling different amino acid residues to NH2-LH2, these probes can be treated by amino peptidyl N (APN, a type II membrane-bound metalloproteinase). Apparently dysregulated in many human malignancies) activation, which produces bioluminescence in the presence of Fuc.

Figure 1. Caged luciferins for detection of enzyme activity in vivo. (Li S,2021)

SPR and BLI Usage Scenarios

SPR and BLI, as two mainstream labeling-free biomolecular interaction analysis techniques, have their own unique application scenarios. Which technique to choose depends mainly on the research objective, sample type, and experimental needs.

Table 2. How to choose SPR and BLI

Because of its high sensitivity and real-time monitoring capability, SPR technology is particularly suitable for research scenarios requiring in-depth molecular mechanisms. For example, in drug screening and development, SPR can monitor the binding dynamics of drug molecules with target proteins in real-time, providing accurate binding rates, dissociation rates, affinity, and data to help researchers optimize drug design. In addition, SPR also performs well in the study of protein interaction and can detect the binding affinity between proteins with high accuracy, which is suitable for the study of complex interactions such as antibody-antigen, receptor-ligand, and so on. For the detection of low-concentration samples, SPR is an irreplaceable choice, because its sensitivity is far greater than BLI. If the study requires high-quality combined kinetic and thermodynamic data, SPR is undoubtedly a better choice.

In contrast, BLI technology is more suitable for high-throughput screening and rapid detection scenarios. Because of its easy operation and fast experimental speed, BLI is often used in large-scale screening experiments, such as antibody library screening or preliminary binding experiments of small molecule compound libraries. The BLI eliminates the need for a complex fluid system and allows the sample to be directly immersed in the sensor, making it ideal for situations where fast results are needed. In addition, BLI's equipment costs are lower, making it suitable for research teams with limited budgets. However, the sensitivity and data accuracy of BLI are relatively low, which makes it more suitable for preliminary screening or qualitative analysis than for in-depth studies requiring high-precision dynamic data.