Elsevier

Analytical Biochemistry

Volume 460, 1 September 2014, Pages 9-15
Analytical Biochemistry

Equilibrium and dynamic design principles for binding molecules engineered for reagentless biosensors

https://doi.org/10.1016/j.ab.2014.04.036Get rights and content

Abstract

Reagentless biosensors rely on the interaction of a binding partner and its target to generate a change in fluorescent signal using an environment-sensitive fluorophore or Förster resonance energy transfer. Binding affinity can exert a significant influence on both the equilibrium and the dynamic response characteristics of such a biosensor. We here develop a kinetic model for the dynamic performance of a reagentless biosensor. Using a sinusoidal signal for ligand concentration, our findings suggest that it is optimal to use a binding moiety whose equilibrium dissociation constant matches that of the average predicted input signal, while maximizing both the association rate constant and the dissociation rate constant at the necessary ratio to create the desired equilibrium constant. Although practical limitations constrain the attainment of these objectives, the derivation of these design principles provides guidance for improved reagentless biosensor performance and metrics for quality standards in the development of biosensors. These concepts are broadly relevant to reagentless biosensor modalities.

Section snippets

Model formulation

The system consists of three state variables: the concentrations of ligand (L), unbound sensor (SF) and bound sensor (SB). By virtue of mass balance, the sum of the concentration of unbound and bound sensor is always equal to the total sensor concentration constant (STot). A linear correlation between the bound sensor and the output signal intensity is assumed. The two rate constants governing this process are the association (kon) and the dissociation (koff) rate constant.

The mathematical

Dynamic consideration reveals the crucial importance of kinetic rates optimization

Intuitively, a sensor that has a very high affinity for its ligand might be expected to perform as a weak dynamic sensor since the characteristic time for complex dissociation would likely be much greater than the period of the signal. Relevant input signal conditions depend greatly on the system under study. In Fig. 1 we show approximate concentrations and time scales for concentration variation for various classes of biological events. Many physiological processes result in great variation of

Discussion

We have shown that there exists an optimal combination of the design parameters kon and koff for a reagentless biosensor and that these vary depending on the nature of the signal. What our results indicate is that the careful determination of binding kinetics is crucial for successful application of biosensors. As a general rule, the KD of the interaction must match that of the expected mean ligand concentration to ensure greatest sensitivity. Biosensors with a KD lower that the mean ligand

Acknowledgments

The authors thank Dr. Alan Wells and Dr. Neda Bagheri for helpful discussions. This work was supported by the Integrative Cancer Biology Program (ICBP 1 U54 CA112967) and by NIH R01 EB 010246.

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