Why is small molecule analysis important?
In pharmacology and molecular biology, “small molecules” have a molecular mass below around 900 Daltons.1 This distinction is far from arbitrary: The smaller a molecule is, the more easily it can permeate living systems. Small molecules are better able to diffuse across cell membranes, enabling them to reach intracellular action sites and giving them generally much higher bioavailability than larger “macromolecules” such as RNA or proteins.2
For this reason, the vast majority of pharmaceutical drugs are “small” molecules (with a few notable exceptions, such as insulin, which is a protein and consequently classed as a macromolecule).
Small molecule analysis is therefore a key concern of pharmacology and molecular biology. In this context, “small molecule analysis” generally refers not only to the identification and quantification of small molecules themselves but to the analysis of their interactions with other molecules of interest (such as receptors). Yielding information on concentration, kinetics, binding and interaction affinities, small molecule analysis is fundamental to drug discovery and biomolecular research.
What is biolayer interferometry and how does it work?
First developed in the early 21st century, biolayer interferometry (BLI) is a relatively new technique for small molecule analysis.3–5
BLI uses the principle of optical interference to monitor binding activity between a pair of unique biomolecules.6 One of these molecules – the ligand – is bound (immobilized) to the surface of a fiber optic tip which has been coated with a biocompatible matrix to enable selective binding. Once the tip is prepared, it is “dipped” into a solution containing the other molecule in the pair (the analyte).
As the analyte begins to interact with the ligand on the sensor tip, the optical thickness of the biological layer (consisting of ligand and analyte) on the surface of the sensing tip increases. Small molecule analysis is enabled by monitoring the precise thickness of this layer of interacting molecules using white light interferometry, which compares the spectral shift to that of a reference surface.
How can biolayer interferometry be used for small molecule analysis?
BLI provides real-time label-free monitoring of molecular interactions on the sensor surface by continuously measuring the thickness of the layer formed by ligand-analyte interactions. This is why BLI is such a powerful tool for small molecule analysis.
Alongside surface plasmon resonance, BFI is one of only a few widely available biosensing techniques which doesn’t require labeling. However, BFI offers a few unique advantages for small molecule analysis.
Crucially, only molecules binding to or dissociating from the sensor can shift the produced interference pattern and generate a response profile. This means that unbound molecules, changes in flow rate, or changes in the refractive index of the surrounding medium don’t influence measurements. BLI is also fluidics-free and can carry out small molecule analysis for either crude or purified samples.
BLI is sensitive enough for peptide and small molecule analysis; and, thanks to its simple operation and the use of reusable fiber-optic tips, is well-suited to parallelized high-throughput applications.
BLI Small Molecule Analysis from Gator Bio
Founded by the inventors of biolayer interferometry, Gator Bio is a world-leading producer of BLI platforms for small molecule analysis applications.7
Combining a 1 mm glass rod with patented optical layers and specialized biosensor surface chemistry, Gator® BLI systems allow for high-capacity immobilization of biotinylated proteins for a wide range of molecular weights. Following immobilization, Gator® systems enable rapid and accurate small molecule analysis, determining critical interaction parameters such as kon, koff, and kD for molecules down to 150 Da.
Are you interested in finding out more about the Gator BLI platform for small molecule analysis? Get in touch with us today.
References and Further Reading
1. Dougherty, T. J. & Pucci, M. J. Antibiotic Discovery and Development. (Springer Science & Business Media, 2011).
2. Veber, D. F. et al. Molecular Properties That Influence the Oral Bioavailability of Drug Candidates. J. Med. Chem. 45, 2615–2623 (2002).
3. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization – PubMed. https://pubmed.ncbi.nlm.nih.gov/19758119/.
4. Biosensor-based small molecule fragment screening with biolayer interferometry – PubMed. https://pubmed.ncbi.nlm.nih.gov/21660516/.
5. Biolayer Interferometry | Gator Bio. https://www.gatorbio.com/technology.
6. Apiyo, D. O. Chapter 10:Biolayer Interferometry (Octet) for Label-free Biomolecular Interaction Sensing. in Handbook of Surface Plasmon Resonance 356–397 (2017). doi:10.1039/9781788010283-00356.
7. About | Gator Bio. https://www.gatorbio.com/about.