AGILE R100 is a graphene biosensor with unprecedented functionality, providing label-free, real-time kinetic binding data. As a novel technology, AGILE R100 is compared to MicroCal iTC200 to demonstrate the benefits of AGILE technology compared to a standard kinetic binding analysis tool, isothermal titration calorimetry (ITC). In this study, kinetic binding data is compared by looking at the Rho guanosine triphosphate hydrolase enzyme (GTPase) interaction with a GTPase activating protein (GAP). The association and dissociation binding rate (ka and kd, respectively) and dissociation constants (KD) are reported. AGILE R100 has a precise binding affinity measurement (KD = 1.35 ± 0.06 µM), comparable to MicroCal iTC200 (KD = 2.7 ± 0.3 µM), while also obtaining ka and kd values. Additionally, AGILE R100 uses up to 30 million times less sample material and up to 30 times less sample volume and can perform 2.5 times more measurements in a given workday compared to MicroCal iTC200.
Small molecules are beneficial as drug therapeutics but often have low solubility in aqueous solutions. Solvents such as dimethyl sulfoxide (DMSO) are necessary additives for small molecule solubility, but these solvents can interfere with optical biosensing platforms. In this paper, AGILE R100 successfully detects the interaction between the cytokine tumor necrosis factor alpha (TNFα) and the small molecule inhibitor SPD304 in 0%, 1%, 3%, and 10% DMSO. DMSO in buffer is compatible with AGILE R100 biosensors and does not interfere with AGILE R100’s ability to measure biomolecular interactions. Additionally, high concentrations of SPD304 (100 μM) were detected on AGILE R100. Importantly, AGILE R100 detects the association binding rate (ka) and the dissociation binding rate (kd) and can calculate the dissociation constant (KD) of the TNFα and SPD304 interaction in buffer with DMSO. The KD reported using AGILE R100 (KD = 14.5 ± 1.8 μM in phosphate buffer saline [PBS] with 10% DMSO) is similar to the previously reported KD value (7.3 ± 0.5 μM in citrate phosphate buffer with 10% DMSO).
Biosensors are used in a variety of fields to characterize biomolecular interactions in real-time. The results of these interaction studies inform the development of the next generation of diagnostics and therapeutics and provide insights into the mechanisms by which pathogens evolve and infect. Label-free assays are particularly useful in these endeavors because they eliminate both the chance of the added label interfering with the native binding chemistry and the additional chemistry steps to attach the label to the molecule under investigation. Data collected from a real-time, label-free biosensor can be analyzed using several models, and the optimal model depends on the prevalence of second order interactions in the system. AGILE R100 collects biosensor data that can be analyzed using kinetic binding models developed over the last few decades. Independent extraction of association and dissociation binding rates allows for advanced molecular design for pharmaceutical applications.
AGILE R100 detects the interaction between the cytokine tumor necrosis factor alpha (TNFα) and the small molecule inhibitor SPD304 with high reproducibility between independent replicate measurements. AGILE R100 detects the association binding rate (ka) and the dissociation binding rate (kd) and calculates the dissociation constant (KD) of the TNFα and SPD304 interaction with minimal deviation between independent replicate measurements (CV < 20%).