Fluorescence, luminescence, and radioactive assays typically rely on specialized reagents labeled with detection moieties. Although such assays are widely used and provide highly valuable information, the process of reagent labeling can be difficult and laborious. Furthermore, labels have the potential to introduce artefacts by inactivating the biomolecule of interest or diminishing its capacity to interact. Affording the advantage of significant time-savings through elimination of the need to perform sample labeling, while allowing for direct acquisition of information rather than inferred information from the label used, label-free detection permits analysis of endogenous biology in action without artificial manipulation of the underlying systems.
Surface plasmon resonance
“The main advantage of Biacore™ is that it’s a real-time technique, meaning you see an entire binding event as it happens,” says Paul Belcher, Ph.D., global product strategy manager, Biacore, at GE Healthcare. “This overcomes the limitations of traditional end-point assays like ELISA and has enabled an extremely broad range of applications. In fact Biacore can be used to study anything from ions to small molecules, peptides, antibodies, and vaccines, through to viruses and whole cells.”
The Biacore technology relies on surface plasmon resonance (SPR), measuring changes in refractive index close to a sensor surface. “If, for example, an antibody is attached to the sensor surface, a change in refractive index occurs upon antigen binding,” explains Belcher. “This change is proportional to the number of molecules that bind, so in essence the instrument measures a change in mass. The number of questions you can ask with SPR is vast—how strong is the interaction I’m interested in (affinity)? How fast is that interaction (kinetics)? Where does my molecule bind? How much of my protein is functionally active? How similar is my biotherapeutic to an innovator drug? Importantly, Biacore can be used to study interactions or target classes where biochemical assays struggle, for example protein-protein interactions.”
Cited in over 15,000 journal articles since 2013, the power of Biacore is exemplified in a recent Nature Communications publication within which the technology was used to identify low molecular weight inhibitors of Ras using fragment-based drug discovery approaches. “Ras is an important target in cancer research but can be challenging to work with,” notes Belcher. “Probing it with sub-300da fragments really requires an extremely sensitive technique like SPR.”
Bio-layer interferometry
“While ELISA is dominant within the study of biomolecular interactions, label-free technologies offer more detailed insights and can allow researchers to select optimal candidates earlier in the drug development process,” notes David Apiyo, Ph.D., applications manager at Pall FortéBio. Relying on Bio-Layer Interferometry (BLI), a technique that measures changing interference patterns of white light reflected by two surfaces—a reference surface and a sample surface—on a biosensor, Pall FortéBio’s Octet® systems perform analysis in microplates, processing multiple samples in parallel to greatly enhance throughput.
“A major advantage of BLI is that no microfluidics are required,” says Apiyo. “This makes the system more robust and provides increased flexibility in sample types and assays. Using Octet systems, protein quantitation of 96 samples can be completed in as little as two minutes, a process that can take from four hours to overnight using traditional ELISA.”
Adding that Octet systems are used by researchers to investigate a wide variety of biomolecular interactions, Apiyo observes that the main goal of these studies is usually to determine affinity constants. “Researchers are also using Octet systems to quantify recombinant proteins and perform in-titer measurements during cell-line development or process development,” he says. “In addition, Octet systems are suitable to detect the presence of contaminants during bioprocessing, while by immobilizing whole cells on the biosensors it is possible to monitor various cellular responses.”
Grating-coupled interferometry
Another popular form of label-free detection, grating-coupled interferometry, employs resonant waveguide grating sensors to measure changes in the refractive index of light following interaction with a surface. Audrey Bergeron, applications scientist II at Corning, describes how each biosensor operating within the company’s Epic® label-free screening technology consists of a glass substrate upon which a periodic optical grating is formed. A dielectric waveguide coating covers the grating. “When illuminated with broadband light, the biosensor reflects a specific wavelength that is a sensitive function of the refractive index close to the sensor surface,” she says. “A biochemical binding event or cellular dynamic mass redistribution (DMR) response leads to mass changes within the detection zone, causing a shift in the resonant wavelength which our system reports as a numerical value.”
“The biosensors used in our Epic technology can detect mass changes within approximately 150 nm of the sensor surface,” elaborates Lynsey Willetts, business director, cell culture at Corning. “They can easily be embedded in a microplate format to enable medium- and high-throughput assays in 96, 384, or 1536 wells. When used in conjunction with our benchtop platform, the Corning Epic BT Reader, an entire plate can be read simultaneously with an update rate of just three seconds.” Willetts adds that since different treatments can be applied to the surface of resonant waveguide grating sensors, applications of Epic technology encompass both biochemical and cell-based assays. “Corning Epic plates have different surfaces to facilitate a plethora of assays. These include analysis of protein-small molecule interactions, particle aggregation, chemotaxis, stem cell differentiation, and antibody-mediated cellular cytotoxicity to name just a few.”
Image: Corning Epic detection principle.
Cell-based label-free assays
“One method to measure a change in a biological event within cells is to insert foreign DNA encoding a target of interest,” explains Gary Allenby, CSO at Aurelia Bioscience. “However the use of manipulated over-expression systems can saturate the endogenous biology, leading to spurious results.” Pointing out that the use of over-expression systems to identify hit compounds can often detect molecules biased toward a particular mechanism that is not representative of the native environment, Allenby says that researchers should be worried they have been missing valuable hits by not using label-free technologies.
“The advantage of performing measurements within physiological systems is the biological relevance of the compound effects. The compound may not hit the target of interest but it may inhibit the pathway, in which case it is a hit that requires deconvolution—this would never have been identified if the assay were set up just to look at interactions with the over-expressed target. Publications suggest that around 50% of the success of drug discovery has come from phenotypic screens and 50% from target-focused approaches—how many compounds may have been missed by the latter?”
Aurelia Bioscience use Corning’s Epic technology for cell-based label-free detection since the system can detect rapid, membrane-based events in either a partial or complete monolayer or, importantly, non-adherent cells. “We typically work with non-adherent immune cells, for example performing studies to measure ligand binding to cell membrane receptors and to evaluate subsequent secondary messenger events,” adds Allenby.
Future development
According to Apiyo, the main limitation of some label-free detection technologies is that one molecule must be immobilized onto a solid surface—a process that may inactivate the molecule. Allenby adds that although label-free adoption for cell-based assays has been slow, approaches measuring resistance have now found a niche within cardio-safety. With awareness of the utility of label-free detection growing, manufacturers continue to push the boundaries of sensitivity and throughput. “Biacore is now used routinely all the way from basic research through to drug discovery and development and into QC and manufacturing,” says Belcher. “This provides a clear indication that label-free detection is here to stay.”