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 graphene biosensor 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
FEB Surface Chemistries
Our FEB biosensor chips have a graphene surface with sensor-specific chemistry, gated by two platinum electrodes that enable and measure electricity flow.
- Graphene is both biocompatible and conductive.
- Binding surface is 2D and highly sensitive.
- Chip material is non-reactive with biomolecules.
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.
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. On optical platforms, background noise caused by optically-dense materials can be magnitudes larger than the signal from target and compound interactions, and must be accounted for by time-consuming and often error-prone solvent correction. In contrast, an FEB system only measures changes in biosensor conductance caused by the energy change in the binding pair when interactions occur. Because FEB is an electrical technique, it registers zero optical background noise and is impartial to optical impediments such as detergents, solvents, or lysates. Agile R100 has detected 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 AND LOW TARGET DENSITY – 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 with low volumes of sample, 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 noise 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. For more details, see Feature Highlight: Sense in Complex Samples: Solvents & Detergents.
NO FLUIDICS – When running label-free kinetic characterization experiments, mass transport effects can skew rate calculations. Surface plasmon resonance (SPR) instruments are particularly affected because they require 100 pg per square mm of immobilized target on the sensor surface to observe reproducible responses. With higher levels of target immobilization comes a greater possibility that the analyte binding rate exceeds the rate at which analyte can reach the sensor surface where the measurement occurs. This mass transport effect makes the observed on-rate slower than the true on-rate. The flip side is also true: High concentrations of immobilized target increase the probability that dissociated analyte will rebind to unoccupied target before it can diffuse out of the matrix. This leads to an observed off-rate slower than the true off-rate. SPR instruments use fluidics to mitigate these mass transport effects, but flow cells add cost, experimental variables, and process complexity.
In contrast, due to the high sensitivity of graphene biosensors, Field Effect Biosensing (FEB) requires less than 1 pg per square mm of immobilized target for accurate data, 100 times less than SPR! Mass transport effects are negated because analyte is pipetted directly onto the sensor surface, which allows the true on-rate to be immediately measured, with no dependence on flow. Because the biosensor surface has a much lower concentration of target, the distance between binders alone is sufficient to allow analyte to diffuse back into solution rather than rebind to immobilized target, enabling the true off-rate to be defined. As a bonus, removing flow cells also reduces the material cost and complications associated with fluidics.
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.