Implications of Specific Buffer Conditions Requirements in Molecular Interaction Studies – and a Method to prevent them

Published Date:
May 22, 2024
Maja Wasilczyk, Brian Sørensen
FIDA Basics

Buffer optimisation is a critical and sometimes time-intensive component of setting up for instance SPR experiments. It easily takes days and sometimes weeks to get the right buffer compositions, pH levels, ionic strengths, additives (like detergents or reducing agents) and temperature. The nature of, e.g. SPR technology requires that the buffer composition and the conditions under which the assay is run are carefully optimised to ensure accurate and meaningful results. Worst case, buffer requirements may indeed limit or prevent the feasibility of running specific assays. But this might change shortly.

Does removing the surface remove the buffer-related issues?  

Same of the buffer optimisation requirements are directly linked to the nature of immobilisation-based technologies, incl. the contraction of non-specific binding, i.e. the attachment of molecules to the sensor chip that are not part of the intended interaction study, avoidance of sensor chip damage and interference with the optical measurements. With in-solution technologies there are significant less buffer optimisation challenges. How come? For example, in In Flow Induced Dispersion Analysis, the measurements are conducted in a free-solution environment, where molecules are not anchored to any solid surface. Since the method measures the diffusion of molecules as they move along the flow path, focusing on changes in their hydrodynamic radius, it is free from surface-related buffer constraints.

Indifference to ionic strength

With no surface to influence binding dynamics, the effect of ionic strength on electrostatic interactions between molecules in solution is less pronounced. The molecules are free to interact with each other without the additional variable of surface charge effects. Furthermore, thanks to uniform flow conditions, molecules are consistently distributed across the capillary's cross-section, reducing the variability in molecular interactions induced by varying ionic strengths.

pH Flexibility

In technologies where biomolecules are immobilised, the pH of the running buffer is a critical parameter, influencing several aspects of the interaction between the analyte and the ligand. This may complicate the interpretation of binding events and necessitate stringent control over buffer pH. On the contrary, in solution-based technologies, such as FIDA, where the molecules are not bound to any surface, their natural charge states are preserved, and their interactions can reflect their true behaviour under physiological conditions. This aspect is critical for accurate bio-molecular studies. Furthermore, since the primary readout in FIDA is diffusion-based, it is inherently less sensitive to pH changes than surface-bound states. The diffusion of molecules in a controlled flow is more indicative of their natural state and less likely to be affected by pH-induced charge variations.

What is the impact on complex biological samples?

You can obtain reliable and meaningful data from immobilisation-based experiments conducted in complex matrices, but it is challenging and requires that multiple parameters are controlled rigorously, incl. sample preparation, non-specific binding, viscosity and mass transport, amongst others. The technology of FIDA provides a more natural and less perturbed environment for studying molecular interactions. By avoiding the complexities introduced by surface immobilisation, FIDA provides a clearer and more direct understanding of bio-molecular behaviours. This makes it exceptionally suitable for complex biological samples where control over ionic strength and pH can be challenging, thus broadening the scope of its application in biophysical and pharmaceutical research.

With having to control an array of parameters, the FIDA principle ensures that FIDA can deliver consistent and reliable results across a range of buffer conditions, thereby enhancing its utility as a versatile tool in the field of molecular interaction analysis.

What about detergents?

The purpose of detergents is to either solubilise or stabilise the protein of interest. FIDA relies on observing changes in dispersion patterns, i.e. the physical properties like size and shape rather than chemical properties altered by detergents. Furthermore, with no surface interaction, detergents do not alter the fundamental measurements of molecular size and interactions.

The ability to use detergents without compromising assay integrity expands  users’ research capacities to a wider range of biological samples. This includes complex mixtures where detergents might be used to disrupt protein complexes or to solubilise certain components. For instance, for membrane proteins, which require detergents for proper solubilisation and to mimic the lipid bilayer environment, FIDA allows direct studies in their native-like states facilitated by detergents.

How does it impact day-to-day research?

Working effectively across a broad range of buffer conditions, including variations in ionic strength, pH, and detergents, enable researchers to analyse samples closer to their physiological conditions, and todo so in a way whereby it is you and the research project that sets the conditions of the experiment and not the limitation of the technology. Summing it up, working in-solution provides the following advantages concerning buffers.

- Consistency issues: (buffer control can be time consuming and error-prone), whereby small deviations can lead to significant variations in results, making reproducibility a major concern

- Resource intensity: Time, reagents, workforce needed to prepare and validate buffer conditions increases complexity and cost

- Limitations on scalability: stringent buffer requirements make later-stage scaling cumbersome

- Emotional workload: patience needed in the process of buffer optimisation, and control increases emotional workload of the researchers engaged. This can be especially impactful on the results-oriented individuals.

In sum, acquiring a methodological independence from surface binding phenomena and allows it to remove buffer-reated workload and expand research capacities. If you would like to test your samples on surface-free technology, sign up for a FIDA pilot here.  

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