Technical Tip: Regeneration of Biosensors in Kinetic Applications

Krista Witte, Ph.D., Sr. Director of Chemistry and Consumables R&D

In most kinetic applications, ease of use and rapid turnaround of data are highly desirable and therefore the single-use biosensor is ideal. However, a subset of applications—for example kinetic screening or epitope binning—can benefit from regenerating and reusing the prepared biosensor. Regeneration can be performed on the Streptavidin (SA) biosensor, the Amine Reactive (AR) biosensor and the Anti-human IgG Fc Capture (AHC) biosensor.

Regeneration of a biosensor refers to using a quick reagent “shock” to disrupt the protein-protein interactions on the sensor. By disrupting the interactions, the protein(s) that were bound to the sensor surface during the screening experiment can be removed and the sensor reused for the next protein of interest. For SA and AR biosensors this means the sensor is regenerated back to the protein that was immobilized on the original surface, the biotin-capture or covalently immobilized capture respectively. For the AHC biosensor this means the biosensor is regenerated all the way back to the original anti-human IgG Fc surface.

The number of times a biosensor can be regenerated and the efficiency and identity of the regeneration reagent greatly depends on the protein-protein interactions being disrupted. Typically interactions can be efficiently disrupted using low pH, high pH, high salt or high detergent concentrations. For most interactions the optimal regeneration conditions must be determined empirically. This Technical Tip outlines a method for determining and validating regeneration conditions.

Regeneration condition development and validation

1. Set up an experiment with the following assay steps:
1. Immobilize first binding partner to the biosensors.
2. Baseline in buffer.
3. Associate second binding partner.
4. Dissociation in buffer.
5. Regeneration (5–30 seconds).
6. Buffer wash (5–30 seconds).
7. Repeat steps E and F 2–4 times more.
8. After regeneration, rebind. For AR and SA rebind second binding partner. For AHC reload the Fc protein and rebind second protein.
9. Repeat steps D–G for the number of cycles to be validated (typically one more cycle than expected in the final experiment).
2. If the regeneration conditions are unknown, the assay plate can be set up to screen up to eight conditions simultaneously. A separate biosensor will be needed for each condition to be tested. The plate example shown in Figure 1 was used to screen eight regeneration conditions for a receptor-protein interaction using eight SA biosensors.
3. Place sample plate and biosensor into the instrument and run the assay. Figure 2 shows an example of a regeneration scouting assay.
4. Once the experiment has been completed, the efficiency of the regeneration conditions can be determined by analyzing the total amount of regenerated capacity on the sensor for the second binding protein over the entire assay run. An example of this analysis is shown in Figure 3.
1. The best conditions show the smallest decrease in binding over the repeated binding/regeneration cycles.
2. In some cases, binding capacity may decrease during the first regeneration cycle but the capacity stabilizes for the subsequent cycles. This can be due to many factors including surface rearrangement or the loss of a small amount of immobilized protein during the first exposure to the regeneration conditions. To minimize the effect of this initial adjustment on the final data, it is common to run an initial binding/regeneration cycle to condition the surface to the regeneration reagent. Data from this first cycle is not analyzed in the final analysis.

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