274239 Controlling the Density of Binding Sites On Silica Based Biosensors
The ideal label-free optical biosensor will be both specific and sensitive for a given target molecule. The sensitivity is governed by the inherent device properties, such as optical loss or photon lifetime whereas the specificity is determined by the accuracy of the surface functionalization employed. Over the past decade, significant advances have been made in device engineering and optimization, resulting in numerous high sensitivity devices. However, complementary research into methods to effectively functionalize surfaces and manipulate functionalized surfaces has not kept pace.
Recently, a method for covalently attaching targeting molecules to optical resonant cavities was developed. Using this approach, it was possible to both maintain the sensing performance of the cavity and form dense, uniform layers of binding sites. However, a dense layer is not always desired, as the binding sites can interfere with each other. Additionally, the number of binding sites on the surface plays a role in determining the overall sensor detection threshold and detection time. Therefore, by developing methods to tune the density of the binding sites, it will be possible to change the device performance.
In the present work, the probe density is adjusted by introducing a secondary “blocker” molecule, where biotin is the probe and a PEG chain is the blocker. In this way, the binding layer is still uniform and dense, but the number of active sites within the layer is decreased. As an extension, a mixture of two targeting molecules are used, where each molecule has a different affinity for the same molecule. In this slightly more complex work, HABA (2-hydroxyazobenzene), a biotin analog, is known to have a slightly lower affinity for streptavidin. Therefore, we expect biotin to be capable of displacing HABA in a competitive binding assay.
An NHS-ester chemistry is used to attach the molecules to the devices and the control substrates1. To verify and optimize the changes in binding site density, both the biotin and the HABA are fluorescently labeled. The resulting surfaces are characterized using fluorescence microscopy, and the changes in fluorescence intensity are measured. This change indicates the proportion of HABA:biotin or PEG:biotin. Ongoing work is exploring the performance (detection limit, equilibrium time) as a function of binding site density.
1. H. K. Hunt, C. Soteropulos, and A. M. Armani, “Bioconjugation Strategies for Microtoroidal Optical Resonators,” Sensors. 10, 9317-9336 (2010).