How does Field Effect Biosensing (FEB) work?
Field Effect Biosensing is a breakthrough label-free technology for measuring biomolecular interactions, and it’s different from anything you’ve heard of before. It is an electrical technique that measures the current across a graphene biosensor surface functionalized with immobilized biomolecular targets (Figure 1). Any interaction or binding that occurs on the surface causes a change in conductance of the biosensor (Figure 2) that is monitored in real-time, enabling accurate kinetic, affinity, and concentration measurements. FEB is a unique orthogonal technology that works when optical methods fail, and it can only be found with innovative personal benchtop assay Agile R100.
What are the key benefits of FEB?
- Label-free detection with real-time results
- Complex sample compatibility, including detergents, solvents, cell fractions, and tissue lysate
- Measurement of molecules >10 Da, from fragments and small molecules to proteins and antibodies.
- Unprecedented sensitivity with an 11-log dynamic range
- Just one 10 µL drop of sample, reducing your cost to data
- Reliable, accurate kinetic characterization at a price every lab can afford
How is FEB data displayed?
Agile R100, an FEB system, lets you monitor the interaction between two molecules in real-time. One is immobilized to the biosensor surface, and the other is a free-in-solution sample applied directly to the surface via a pipettor. The sensorgram to the right plots the response versus time, and is typical of how a real-time measurement is represented with the Agile R100 system and software. The magnitude and shape of the response curve is related to the number of binding events at the biosensor surface and is measured in biosensing units (BU). A BU is the percent change in conductance measured by the system, multiplied by 10.
First, the calibration read in buffer is shown. Then, you can view the immobilization of the target on the biosensor surface, and the association response as added analyte binds to the target. Off-rates can be viewed as the interaction is reversed and dissociation occurs.
Small Molecule Detection with an FEB system
The FEB method on which Agile R100 is based is fundamentally different from optical techniques. Optical tools such as SPR and BLI systems measure mass-dependent shifts in light, which is adequate for large molecules. However, SPR and BLI platforms struggle to measure small molecule interactions because small molecules elicit correspondingly small sensor responses. To find the needle of signal generated in a large haystack of background noise, time-consuming and error-prone solvent correction is required.
In contrast, FEB is a completely orthogonal, breakthrough technology. It is an electrical technique, not an optical, mass-based method. The size of the molecule being measured is irrelevant on an FEB platform because molecule size doesn’t impact what FEB measures: the change in biosensor conductance caused by a binding interaction at its surface. In fact, small molecules generally create optimal effects on an FEB platform because small molecules have large chemical potential shifts in relation to their volume, which produces a large response with FEB. This makes FEB an excellent orthogonal technique for small molecule and fragment characterization and validation.
|No microfluidics - quickly and easily measure unstable proteins|
|Use small volumes - just a 10 µL drop of sample lets you drastically reduce your cost to data|
|Sense in complex samples without error-prone, time-consuming solvent correction|
|11-log dynamic range - develop weakly-binding fragments into high-affinity compounds on one platform|
|Reliable small molecule measurements (> 10 Da) for flexible drug development unlimited by molecule size|
|Small assay system - tuck it in your pocket or put it on your personal benchtop|
HOW FEB AND AGILE R100 SUPPORT NEW CAPABILITIES
SENSE IN COMPLEX SAMPLES – Agile R100 is based on an electrical technique, not an optical one, so it doesn’t have the problems with complex samples that optical biosensors have. Optically-dense materials such as detergents, solvents, or lysates cause large amounts of background noise on SPR or BLI systems, requiring time-consuming and error-prone solvent correction processes. Agile R100 easily circumvents this because an FEB system only measures changes in biosensor conductance caused by the energy change in the binding pair when interactions occur. Unbound molecule mass does not change the conductance of the sensor, therefore, the molecule mass that can obscure optical measurements does not register on the Agile R100. Agile R100 has measured in human plasma, cell lysate, and tissue lysate. Learn more in this poster titled Agile Sensors Quantify Interactions in Challenging Samples for Drug Discovery, presented in 2016.
11-LOG DYNAMIC RANGE – Agile biosensor chips are made with the “super-material” graphene, which gives the platform its unique ability to sense with an unprecedented 11-log dynamic range. Graphene is a two-dimensional material that is one-million times thinner than a human hair, making it the thinnest material on earth.
Because graphene is so thin, every atom of the biosensor surface is exposed to your sample, enabling extreme sensitivity to the changes in conductance measured by an FEB system. Agile R100 can detect even with weak interactions and low concentrations, letting you to develop weakly-binding fragments into high-affinity compounds with accuracy and reliability, on a single platform.
USE SMALL VOLUMES – Single-sample format Agile R100 allows you to pipette sample directly onto the surface of the biosensor chip. Because the biosensor is so small, you only need a 10 uL drop of sample to gain valuable kinetic binding data, helping you preserve your precious sample. Agile R100 is very sensitive, which means you don’t need to coat the biosensor surface with excess target; only a few thousand molecules are needed to functionalize the chip. That means you can use just nM concentrations of target, at low volumes, drastically reducing your cost to data. Visit Data for example experiments using small sample sizes.
SAVE TIME WITH RAPID MEASUREMENTS – Agile R100 saves time in multiple ways. Your sample is applied directly to the sensor surface, which eliminates the need to learn complicated system components and processes. With no microfluidics, you can sense within minutes of sample prep, which lets you measure unstable proteins quickly and easily. Agile R100 lets you view your data in real-time for immediate visualization of results as they occur. This, combined with Agile R100’s single-sample format, enables mid-experiment changes to protocol that shortcuts assay development time and is unfeasible on other label-free platforms.
SENSE IN HIGH CONCENTRATIONS – Small molecule measurements often require high concentrations because interactions have KD values in the µM to mM range. For measurements using such high concentrations of small molecules, it is often necessary to include DMSO in the solution to maintain a known concentration and prevent precipitation. The addition of DMSO has a large effect on the optical properties of a solution. On an optical platform such as SPR or BLI, a 1% difference in DMSO concentration causes background noise that is ten times larger than the binding signal. This unwanted background nose drowns out your binding response and necessitates additional solvent correction measurements. Agile R100 is based on FEB, an electrical technique, not an optical one, so it sidesteps these optical limitations. Learn more about Detecting Interactions of Small Molecules in 10% DMSO Using Agile R100, or see our publication on FEB Increases Signal-to-Noise by 10 Times versus BLI and ELISA.
FITS ON YOUR BENCHTOP – Agile R100 is the kinetic binding platform that you can tuck in your pocket. The FEB technology on which Agile R100 is based enables the system to have a completely breakthrough form factor that is surprisingly compact. Comprised of solely small, portable components, Agile R100 has no optical elements that cause other systems to be bulky and require calibration.
FEB Increases Signal-to-Noise by 10 Times versus BLI and ELISA
Agile R100, a Field Effect Biosensing (FEB) system, detected Zika viral antigen at a concentration 10 times lower than on a BLI system in this breakthrough study. The platform also lowered the limit of detection by 10 times versus an ELISA for inflammatory protein IL-6.