In-solution kinetics by Flow-Induced Dispersion Analysis (FIDA)

A new method that helps overcome the limitations of surface-based kinetics.

Surface-based methods such as Surface Plasmon Resonance (SPR) are a cornerstone in the study of biomolecular interactions, offering real-time insights into the kinetics of these complex processes. At the same time, getting correct data using surface immobilization technologies introduces several challenges; multiple parameters have to be managed and balanced. There are several pitfalls, such as non-specific binding, mass transport limitations, surface heterogeneity, regeneration efficiency issues, and complications arising from fixed orientation upon immobilization, hydrogel length, surface charge, and avidity effects.

These factors can significantly affect the accuracy and reliability of kinetic measurements. Luckily, an emerging orthogonal solution kinetic methodology can now address these shortcomings. Flow-Induced Dispersion Analysis (FIDA) is a technique that offers a powerful approach to studying biomolecular kinetics without the constraints of surface-based methods.

In-Solution Kinetics: The Missing Piece

Non-immobilization kinetics techniques, like FIDA, measure the interactions of biomolecules directly in a flowing solution, bypassing the need for immobilization on a sensor surface. This approach inherently avoids the artefacts associated with surface binding and allows for the observation of biomolecular interactions in a more natural, physiological environment. The essence of FIDA and similar techniques lies in their ability to analyze the diffusion behaviour of molecules as they interact, leveraging the flow-induced dispersion phenomena to extract kinetic and affinity data from the same experimental setup.

Addressing the Challenges of SPR

1. Non-specific Binding: A significant challenge in SPR is distinguishing between specific and non-specific binding to the sensor surface. As underlined by Frutiger et al. (2021, p.1) ‘’Artificial systems such as biosensors that rely on distinguishing specific molecular binding events in a sea of nonspecific interactions have struggled to overcome this issue. Despite the numerous technological advancements in biosensor technologies, nonspecific binding has remained a critical bottleneck due to the lack of a fundamental understanding of the phenomenon. In-solution kinetics circumvents this issue entirely by analyzing the molecules in physiologically relevant conditions without the artificial context of a surface, thus eliminating false positives or negatives arising from non-specific binding.
2. Mass Transport Limitations: SPR measurements can be skewed by the rate at which analytes diffuse to the sensor surface (Sohuok & Zhao, 2010), particularly at high concentrations or under slow flow conditions. This can be compounded by the functional heterogeneity of surface binding sites, which may be intrinsically inhomogeneous or rendered heterogeneous by the immobilization process, affecting the binding properties and complicating the interpretation of kinetic data. Additionally, the presence of mass transport limitation can introduce artefacts in the measured kinetics (Svitel et al., 2007). Fortunately, thanks to the fact that FIDA measures interactions as they occur homogeneously in solution, it is insensitive to mass transport effects that plague surface-based assays and avoids the complexities introduced by heterogeneous immobilization of binding partners.
3. Surface Heterogeneity and Orientation: The variability in ligand density and activity across an SPR sensor chip, along with the fixed orientation of immobilized molecules, can complicate data interpretation and require complex analytical methods (see e.g. Gaudreault et al., 2021). In-solution kinetics methods do not suffer from these issues, as there is no immobilization or heterogeneous surface to influence the interaction dynamics, and the molecules are free to rotate, providing a more natural interaction scenario.
4. Regeneration Efficiency and Hydrogel Thickness: SPR requires the sensor surface to be regenerated between experiments, a process that can damage the ligand or leave residual analyte, affecting subsequent measurements. The measurements in SPR are also influenced by hydrogel thickness; a thicker hydrogel layer increases the distance between the sensor surface and the medium, reduces the penetration depth and leads to reduced sensitivity. FIDA and other in-solution approaches eliminate the need for regeneration, allowing for more consistent and repeatable measurements, and are not affected by such spatial constraints.
5. Surface Charge and Avidity: The highly negatively charged SPR surface can distort affinity measurements if the interacting molecules carry a net charge. Additionally, the physical proximity of immobilized proteins to the sensor surface can alter binding affinities due to spatial constraints and surface interactions (Olmsted et al., 2016). Furthermore, surface-based measurements can be susceptible to avidity artefacts, such as 'bridging' seen in polyubiquitin-binding assays, which can lead to overestimated binding affinities for certain chain types (Schoeffler et al., 2021). Such effects of surface immobilization highlight the advantage of in-solution kinetics, like FIDA, which are not influenced by surface charges and avoid complications related to avidity by analyzing molecules in their freely diffusing state.

The Advantages of Non-Immobilization Method - FIDA

FIDA specifically utilizes the principle that the diffusion of molecules in a laminar flow will vary depending on their size and interactions. By observing how molecules disperse in a controlled flow environment, FIDA can measure fraction bound based on their diffusion coefficients. This enables the direct measurement of binding kinetics and affinities without the artefacts associated with surface attachment. Furthermore, FIDA can be applied to a wide range of molecular sizes and types, from small drug molecules to large protein complexes, offering versatility that complements and, in some cases, surpasses traditional SPR capabilities.

One crucial added advantage is the full control over buffer composition (e.g., saltiness) and other environmental factors, including temperature. Aggregation is also not an issue, and it can additionally be quantified through an embedded QC Module. Moreover, FIDA detects oligomerisation and conformational changes, as it measures molecular size, not just weight, allowing for the observation of oligomerization and conformational changes in the interacting biomolecules.

The Future of Biomolecular Interaction Analysis

The move towards in-solution kinetic analysis represents a significant advancement in the field of biomolecular research. Techniques like FIDA not only overcome the limitations of traditional surface-based methods; they also provide a more accurate reflection of biological systems. This shift is poised to enhance our understanding of biomolecular interactions, with profound implications for drug discovery, molecular biology, and beyond.

In sum, while surface-based technologies are invaluable in the study of biomolecular kinetics, the advent of in-solution techniques like FIDA offers a promising alternative that addresses many of their inherent limitations, such as issues with fixed molecular orientation, hydrogel-related artefacts, surface charge influences, and avidity biases. By enabling more accurate, reliable, and physiologically relevant measurements, including the detection of conformational changes, in-solution kinetics are setting a new standard for the analysis of biomolecular interactions.

References:?

Frutiger, A., Tanno, A., Hwu, S., Tiefenauer, R. F., V?r?s, J., & Nakatsuka, N. (2021). Nonspecific Binding-Fundamental Concepts and Consequences for Biosensing Applications.?Chemical reviews,?121(13), 8095–8160. https://doi.org/10.1021/acs.chemrev.1c00044

Gaudreault, J., Forest-Nault, C., De Crescenzo, G., Durocher, Y., & Henry, O. (2021). On the Use of Surface Plasmon Resonance-Based Biosensors for Advanced Bioprocess Monitoring. Processes, 9(11), 1996. https://doi.org/10.3390/pr9111996

Olmsted, I. R., Kussrow, A., & Bornhop, D. J. (2012). Comparison of free-solution and surface-immobilized molecular interactions using a single platform.?Analytical chemistry,?84(24), 10817–10822. https://doi.org/10.1021/ac302933h

Schuck, P., & Zhao, H. (2010). The role of mass transport limitation and surface heterogeneity in the biophysical characterization of macromolecular binding processes by SPR biosensing.?Methods in molecular biology (Clifton, N.J.),?627, 15–54. https://doi.org/10.1007/978-1-60761-670-2_2

Schoeffler, A. J., Helgason, E., Popovych, N., & Dueber, E. C. (2021). Diagnosing and mitigating method-based avidity artifacts that confound polyubiquitin-binding assays.?Biophysical reports,?1(2), 100033. https://doi.org/10.1016/j.bpr.2021.100033

?Svitel, J., Boukari, H., Van Ryk, D., Willson, R. C., & Schuck, P. (2007). Probing the functional heterogeneity of surface binding sites by analysis of experimental binding traces and the effect of mass transport limitation.?Biophysical journal,?92(5), 1742–1758. https://doi.org/10.1529/biophysj.106.094615