Department of Biological Engineering, University of Missouri
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Soteropulos, C. E., Hunt, H. K. Attaching Biological Probes to Silica Optical Biosensors Using Silane Coupling Agents. J. Vis. Exp. (63), e3866, doi:10.3791/3866 (2012).
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.
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 as an explanation of our motivation for choosing these techniques.8
The success of these reactions can be evaluated through optical and fluorescence microscopy after the addition of the primary amine, as well as after the addition of the biotin. While optical microscopy can be used to determine if the surface functionalization protocols resulted in damage to the microsphere surface, or even contamination, fluorescence microscopy is used to verify the quality and uniformity of surface coverage of the biotin molecule, as well as the capability of the biotin on the surface to bind with (strept)avidin. To evaluate the coverage of amines on the surface, we used fluorescein isothiocyanate (FITC) fluorescent dye, which reacts with primary amines to form stable, covalent thiourea linkage to the microsphere. Texas Red fluorescent dye that has been conjugated to avidin is used to label the biotin groups on the surface through the biotin-avidin interaction. In both cases, dyes were chosen that can interact (through either receptor-ligand interactions or covalent bonding) with the functional groups on the surface.
Here, the silane coupling agent, which has three leaving groups and one functional group, provides the bridge between the inorganic surface and organic probe molecule. The primary amine functionality reacts quantitatively with NHS esters to form stable amide bonds. In this case, we use a biotin probe molecule whose valeric side chain has been modified with an NHS ester group. Realistically, any probe molecule to which an NHS ester group can be added to the surface using the following protocols. Additionally, these protocols are general, and can be used to functionalize any silica device surface with biotin.
The primary challenge with these protocols is not actually in the chemistry itself, but rather in the handling of the silica microspheres. Please note that, throughout the protocols, the microspheres should be handled by grapsing their stems lightly with sharp-tipped tweezers. This keeps the tweezers well away from the microspheres themselves, and allows for easy transport between steps. Many steps in the protocol below are specifically designed to address this factor.
2. Microsphere Fabrication
3. Populating the Surface with Hydroxyl Groups
4. Attaching Silane Coupling Agents to the Surface
5. Attaching Biotin to the Surface
6. Fluorescent labeling of amine-terminated silica
7. Fluorescent labeling of biotin-terminated silica
8. Representative Results
Correctly functionalized microspheres can be identified through optical and fluorescence microscopy. If the surface functionalization is done correctly, it should result in a uniformly dense coverage of biotin molecules on the surface, and the surface should remain defect- and contaminant-free after functionalization in order to maintain their high sensitivities during detection experiments. Optical microscopy can be used to probe the latter, while fluorescent microscopy can probe the quality and uniformity of surface coverage. In Figure 2, we show examples of correctly functionalized microspheres. These images show that there is no surface damage or contamination due to functionalization (Fig. 2a), and that the microspheres show a uniform, consistent coverage of either amine groups (Fig. 2b) or biotin groups (Fig. 2c) on the surface.
If the microspheres have not been functionalized correctly, the optical microsphere images will exhibit surface contamination, clumping or non-uniform coverage, and obvious defects in the surface, like surface cracks (Figure 3). Here, we see a common example of surface contamination, resulting from clumping of reagents on the surface.
Figure 1. 3-step reaction scheme for attaching probe molecules to the surface of silica microspheres. Click here to view larger figure.
Figure 2. Silica microspheres. a) Optical micrograph of silica microsphere populated with hydroxyl groups via exposure to oxygen plasma; b) Fluorescent micrograph of silica microsphere populated with primary amines and labeled with FITC dye; c) Fluorescent micrograph of silica microsphere populated with biotin and labeled with Texas Red-avidin conjugate. Reprinted with permission from Soteropulos, C. E., Hunt, H. K. & Armani, A. M. Determination of binding kinetics using whispering gallery mode microcavities. Appl. Phys. Lett. 99, 103703-103703 (2011). Copyright 2011, American Institute of Physics.17
Figure 3. Optical micrograph of improperly functionalized sphere. Here, you can see dust on the top right surface, as well as contamination extending off the surface. Additionally, the right side of the optical microsphere shows a small divot on the surface.
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 during the functionalization process. It was hard to avoid brushing the microsphere against an object when moving each individually with tweezers by holding onto the stems. By stabilizing the position of each microsphere on the glass slide, we were able to simply move a number of microspheres in and out of different containers by handling the glass slide, and not the stem of the microsphere. In this way, we were able to functionalize many of these devices at the same time, and we eliminated contamination and surface damage caused by improper handling, resulting in an ~ 90% success rate of creating undamaged, functionalized microspheres.
As stated in the protocols, we explored two methods by which to hydroxylate the silica microspheres. While piranha solution is commonly used for this, it can be time-consuming, and requires wet chemistry. When working with microspheres, we noted the superiority of hydroxylation through oxygen plasma treatment. Oxygen plasma treatment requires no contact with liquid solutions, no drying, and no handling of the silica microspheres. Additionally, it is a much faster process to populate the surface with hydroxyl groups. However, we recognize that not every research group will have access to oxygen plasma equipment, and therefore may need to use piranha solution to form the requisite hydroxyl functionality. The use of either will result in the formation of hydroxyl groups on the surface.
The prevention of contamination during surface functionalization is a major issue, but it can be minimized by careful adherence to the protocols. The most likely causes of contamination are poor handling during functionalization, as well as improper or non-thorough washing of the surface after each of the reaction steps. This is particularly the case during the first reaction step, where the surface is populated with hydroxyl groups. If the piranha solution is chosen for this method, care must be taken to thoroughly remove any sulfuric acid from the surface, and to thoroughly dry the microsphere, as the surface is now very hydrophilic, and water from the rinses clings to the surface. If sulfuric acid remains on the surface, the microsphere becomes "sticky" and dust from the laboratory environment collects on the surface more readily. Additionally, water droplets on the surface cause the silane coupling agent to congregate in the area of the water droplets, forming a polymeric silica glob, which is seen as clumping in the fluorescent micrographs. If surface clumping does occur, it prevents the microsphere from being used in detection experiments, because of a large drop in the sensitivity of the device. Typically, when oxygen plasma treatment is used, surface clumping, as well as contamination, is minimized. However, not every laboratory has access to this equipment, so using the piranha solution may be a necessity.
Finally, we found it was necessary to sonicate the NHS-biotin solution prior to use to fully dissolve the NHS-biotin in DMSO. When this step was skipped, the NHS-biotin would deposit onto the surface in large clumps via physical adsorption, rather than covalently binding to the surface. These large clumps are extremely difficult to remove during the washing steps, but can be prevented by proper dispersal during the initial dissolution. One hour is typically sufficient for this purpose; however, if these procedures are extended to other probe molecules modified with NHS ester groups, we recommend evaluating the solution via dynamic light scattering to ensure full dissolution.
The protocols detailed here represent a step forward in the translation of planar silica surface chemistries to three-dimensional devices, where the quality of the surface coverage and the device surface are equally important for the final use of the devices. When these protocols are used , the fully functionalized microspheres showed uniformly dense coverage of the probe molecule, with no contamination or clumping on the surface. This uniform surface coverage allows for the maintenance of high sensitivities when these devices are used in detection experiments. The use of silane coupling agents as a bridge between the inorganic substrate and the biological probe molecule is simple and straightforward, and can be used, particularly with the well-understood (and very common) NHS ester chemistry, to populate optical biosensor surfaces with probe molecules of various types.
No conflicts of interest declared.
The authors gratefully acknowledge Prof. Andrea Armani at the University of Southern California for support during the time this protocol was developed. Funding for the initial development of this work was provided by the National Science Foundation [085281 and 1028440] and the National Institute of Health through NIH Director's New Innovator Award Program [1DP2OD007391-01]. Additional information is available at http://web.missouri.edu/~hunthk/.