Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents

Summary

Biosensors interface with complex, biological environments and perform targeted detection by combining highly sensitive sensors with highly specific probes attached to the sensor via surface modification. Here, we demonstrate the surface functionalization of silica optical sensors with biotin using silane coupling agents to bridge the sensor and the biological environment.

Abstract

In order to interface with biological environments, biosensor platforms, such as the popular Biacore system (based on the Surface Plasmon Resonance (SPR) technique), make use of various surface modification techniques, that can, for example, prevent surface fouling, tune the hydrophobicity / hydrophilicity of the surface, adapt to a variety of electronic environments, and most frequently, induce specificity towards a target of interest.^1-5 These techniques extend the functionality of otherwise highly sensitive biosensors to real-world applications in complex environments, such as blood, urine, and wastewater analysis.^2,6-7 While commercial biosensing platforms, such as Biacore, have well-understood, standard techniques for performing such surface modifications, these techniques have not been translated in a standardized fashion to other label-free biosensing platforms, such as Whispering Gallery Mode (WGM) optical resonators.^8-9

WGM optical resonators represent a promising technology for performing label-free detection of a wide variety of species at ultra-low concentrations.^6,10-12 The high sensitivity of these platforms is a result of their unique geometric optics: WGM optical resonators confine circulating light at specific, integral resonance frequencies.¹³ Like the SPR platforms, the optical field is not totally confined to the sensor device, but evanesces; this "evanescent tail" can then interact with species in the surrounding environment. This interaction causes the effective refractive index of the optical field to change, resulting in a slight, but detectable, shift in the resonance frequency of the device. Because the optical field circulates, it can interact many times with the environment, resulting in an inherent amplification of the signal, and very high sensitivities to minor changes in the environment.^2,14-15

To perform targeted detection in complex environments, these platforms must be paired with a probe molecule (usually one half of a binding pair, e.g. antibodies / antigens) through surface modification.² Although WGM optical resonators can be fabricated in several geometries from a variety of material systems, the silica microsphere is the most common. These microspheres are generally fabricated on the end of an optical fiber, which provides a "stem" by which the microspheres can be handled during functionalization and detection experiments. Silica surface chemistries may be applied to attach probe molecules to their surfaces; however, traditional techniques generated for planar substrates are often not adequate for these three-dimensional structures, as any changes to the surface of the microspheres (dust, contamination, surface defects, and uneven coatings) can have severe, negative consequences on their detection capabilities. Here, we demonstrate a facile approach for the surface functionalization of silica microsphere WGM optical resonators using silane coupling agents to bridge the inorganic surface and the biological environment, by attaching biotin to the silica surface.^8,16 Although we use silica microsphere WGM resonators as the sensor system in this report, the protocols are general and can be used to functionalize the surface of any silica device with biotin.

Protocol

1. Background

The biotin is attached to the surface of these devices through a simple, three-step process (Figure 1). First, we clean the surface and populate it with hydroxyl groups by exposing the devices to either oxygen plasma or piranha solution. Second, we use vapor deposition to attach the silane coupling agent terminated with a primary amine to the hydroxyl groups through hydrolysis and condensation reaction. Third, we attach biotin to the surface via N-hydroxysuccinimide (NHS) ester chemistry. We direct the interested reader to our previous work for more information about the development of these techniques, as well ....

Discussion

As described in the protocols, we created a housing platform by which to transport the silica microspheres by their stems throughout the functionalization process. This housing platform was created as a solution to the surface contamination and damage that resulted from the microsphere coming into contact with the walls of the various containers used throughout the functionalization process. We realized the main difficulty arose from constantly attaching and detaching individual microspheres to different containers durin.......