Understanding BRIC in FIDA Technology: A Comprehensive Overview
In the intricate world of molecular research, precision isn’t just a goal—it’s a necessity. As a researcher, you might encounter some critical questions: Can I trust my data to reflect true biological conditions? Is there something I am not seeing, and how could I detect weak molecular interactions?
In such a case, a technology which delivers a numerous, complimentary, and simultaneous readouts, while keeping native sample conditions, comes in handy. Flow Induced Dispersion Analysis (FIDA), is such a technology.
FIDA’s primary measurement, the hydrodynamic radius (Rh), offers an exceptionally precise readout of molecular size in solution, capable of detecting changes as small as 5%. This precision is crucial for accurately characterising molecular complexes. Yet, when size changes fall below this threshold, BRIC (Binding Related Intensity Change) becomes invaluable. As a fluorescence-based readout, BRIC sensitively captures binding-induced alterations that may not significantly affect overall size but are critical to understanding molecular interactions. Together, these measurements provide a comprehensive and nuanced view of molecular behaviour, ensuring that no details are overlooked in your research.
What is BRIC?
BRIC stands for Binding Related Intensity Change, and it is an integral aspect of FIDA technology. It measures changes in fluorescence intensity that can occur when molecules (such as proteins or small ligands) bind to each other, or proteins change their conformational state due to environmental changes.
BRIC readout provides information on binding events that might otherwise be difficult to detect, especially in complex biological environments like blood serum or cell lysates.
Importantly for the users, BRIC operates alongside the primary readout of FIDA, the hydrodynamic radius, which offers an absolute measurement of molecular size. Having these two orthogonal readouts provides researchers with more comprehensive and multidimensional understanding of molecular interactions.
Quenching Effects and BRIC
Fluorescence quenching refers to a process where the fluorescence of a molecule (fluorophore) is reduced due to various factors, such as molecular collisions, energy transfer, or changes in the local environment. When a molecular change (e.g. a binding event) occurs, such as a ligand binding to a protein, it can bring the fluorophore into close proximity with quenchers or alter the microenvironment around the fluorophore, leading to changes in fluorescence intensity.
BRIC utilises these changes in fluorescence intensity to detect and quantify binding interactions. Specifically:
- Environmental Changes: Binding interactions often change the polarity, pH, or other characteristics of the fluorophore's microenvironment, which can either enhance or diminish fluorescence. BRIC measures these intensity changes, providing insights into the binding dynamics and molecular interactions.
- Static Quenching: When a binding event causes the formation of a non-fluorescent complex between the fluorophore and a quencher, the fluorescence intensity decreases. This reduction in intensity can be measured by BRIC to indicate that binding has occurred.
In summary, quenching effects are a fundamental aspect of how BRIC detects molecular changes (e.g. binding events). By monitoring the decrease (or increase) in fluorescence intensity caused by quenching, BRIC provides valuable data on the occurrence, strength, and nature of molecular interactions. This makes BRIC a sensitive tool for studying biomolecular interactions in various research and drug development contexts.
The Dual Power of BRIC and Hydrodynamic Radius Readouts
What makes BRIC particularly powerful in FIDA technology is its combination with hydrodynamic radius measurements. The hydrodynamic radius provides an absolute size measurement of the molecules or complexes in solution, offering insights into how binding affects their size and conformation. When paired with the BRIC readout, which detects binding-induced changes in fluorescence, researchers gain a comprehensive understanding of molecular interactions. This dual readout approach is especially valuable in studies where both the size and binding behaviour of molecules are critical to understanding their function.
Binding Event Detection in native conditions: what are the benefits?
A couple of technological advantages of BRIC readout stand out.
Firstly, the readout is automatically adjusted to account for matrix complexity. It is to say, that the signal of interest is recovered and separated from the background signals, allowing to easily work in complex matrices.
Secondly, BRIC signal measurement does not require temperature changes (which are used in other technologies to induce fluorescence changes). It is important to note that in technologies where fluorescence intensity is measured under changing conditions, such as those involving heat, the readout can become unreliable or even useless due to the altered state of the sample. Since in FIDA the temperature is kept constant, this does not occur and conditions remain native.
Thus, on the practical side, using BRIC measurement (as a part of FIDA assay) for has several key benefits:
- Maintenance of Native Conditions: BRIC allows the study of biomolecular interactions under physiological conditions without the risk of denaturing or altering the native structure of the molecules due to temperature shifts. Thus, delivering more accurate and reliable data.
- Stability and Consistency: By controlling for temperature changes, BRIC ensures that the measurements are consistent and not influenced by external factors that could introduce variability, particularly in complex biological samples.
- Compatibility with Complex Media: The ability to measure interactions at constant temperatures, and accounting for background noises makes BRIC especially useful for analysing samples in complex media like blood or serum, where temperature changes could otherwise compromise the integrity of the sample and where samples are of high complexity. (Here, it is crucial to note that FIDA technology is generally highly compatible with complex media and wide array of buffers.)
Applications of FIDA measurements: BRIC, hydrodynamicradius & Lambda Dynamics across research areas
BRIC, in conjunction with other readouts by FIDA technology such as hydrodynamic radius (Rh) and Lambda Dynamics, provide valuable data across a wide range of research areas:
- Drug Discovery and Development: BRIC combined with Rh come in handy in screening potential drug candidates by measuring their binding affinities to target molecules. It also aids in understanding the kinetics of these interactions, which is crucial for assessing the efficacy and safety of new drugs.
- Protein-Protein and Protein-Ligand Interactions: BRIC helps in characterising the interactions between proteins or between proteins and small molecules. This is particularly important in understanding the structural basis of these interactions and how they influence biological functions.
- Lipid Nanoparticles: BRIC is utilised to analyse and quantify the interactions between lipid nanoparticles and target proteins. This application is crucial in the development of targeted drug delivery systems, where precise sizing and interaction data in complex media like serum or plasma ensure the stability, biocompatibility, and effectiveness of LNPs during drug development and manufacturing process
- Enzyme Kinetics and Inhibition Studies: BRIC allows researchers to study how enzymes interact with their substrates or inhibitors, providing insights into the mechanisms of enzyme action and the development of enzyme-targeting drugs.
Wavelength shifts (Lambda Dynamics)
In FIDA, Lambda Dynamics refers to the shifts in the wavelength (lambda) of the detected signal, which occur when binding events change the environment of the fluorophore or other optical markers.These shifts provide additional insights into the binding interactions and can indicate changes in the conformation or orientation of the molecules involved.This readout, combined with BRIC (Binding Related Intensity Change) and hydrodynamic radius measurements, gives researchers a detailed picture of molecular interactions, making FIDA an exceptionally powerful tool for studying complex biological processes, drug interactions, and molecular dynamics in their native environment.
Conclusion
BRIC in FIDA technology represents a significant advancement in the study of biomolecular interactions. Its ability to provide detailed binding information without the need for temperature changes, alongside precise hydrodynamic radius measurements, makes it an indispensable tool in various fields of research. Whether in drug discovery, biomarker validation, or nanotechnology, BRIC offers researchers the accuracy, sensitivity, and versatility needed to push the boundaries of what we can understand about molecular interactions in their native environments.