(203b) Bioconjugation of Optical Microcavities for Label-Free Sensing | AIChE

(203b) Bioconjugation of Optical Microcavities for Label-Free Sensing


Hunt, H. K. - Presenter, University of Southern California
Soteropulos, C. - Presenter, University of Southern California
Armani, A. M. - Presenter, University of Southern California

The development of biosensors with high sensitivity and specificity is of significant interest to the fields of medical diagnostics and environmental monitoring, where rapid and real-time detection of antigens, bacteria, viruses, etc., is necessary. Traditional, labeled sensors, such as fluorescent immunoassays, are not capable of real-time detection, and detect the presence of the fluorescent probe rather than the molecule of interest itself. In the past decade, a new class of sensors consisting of high-performance optical devices has been developed based on whispering gallery microcavities. Initially designed for telecommunications applications, these structures have demonstrated unique capabilities in the biosensing arena primarily due to their very low optical loss. The low optical or material loss of these devices enables ?optical amplification? of otherwise undetectable signals to occur within the structure, improving the signal to noise ratio. This optical loss metric is quantitatively characterized by the quality factor (Q) of the cavity. The two most commonly used microcavities, the microsphere and the microtoroid, have quality factors in excess of 100 million, which corresponds to photon storage times of greater than 100 ns. As a result of their circular geometry, the optical field orbits at the periphery of the device and is not completely confined within the cavity. This creates an evanescent tail which interacts with the environment, enabling detection of chemical or biological species. For example, using silica microcavity devices, researchers have shown label-free detection of protein conformation changes and bacteria, as well as label-free detection of virus and molecules at the single molecule concentration level.1-2

Previous research efforts have focused attention on the development of the sensor itself. However, while the sensitivity of the device is dependent on the low optical loss, specificity is an equally if not more important feature of any sensing platform. Therefore, it is crucial to develop a high density, covalent surface functionalization process which also maintains the optical device's performance metrics. In the case of the whispering gallery mode sensor, the most important parameter is the Q factor of the cavity. Here, we demonstrate a facile method to impart specificity to optical microcavities without adversely impacting their optical performance. We have used the silica ultra-high-Q microtoroid microcavity as the test platform, because it is the only microcavity fabricated on a silicon substrate which has achieved Q factors in excess of 100 million.3 However, the techniques developed are transferable to other optical cavities, such as microrings, microspheres and microcylinders.

In this approach, we selectively functionalize the surface of the silica microtoroids with amine-terminated silane coupling agents of various lengths using two different methods: 1) organic solvent deposition and 2) vapor deposition grafting techniques. The surface is then biotinylated via N-hydroxysuccinimide (NHS) ester chemistry at the amine end of the linker under typical literature conditions. The as-fabricated and surface-modified devices are characterized qualitatively by optical microscopy and scanning electron microscopy as well as quantitatively by microcavity analysis techniques (Q factor) to determine the impact of the surface functionalization methods on the device sensitivity, and to evaluate the conditions best suited to ensuring the devices' performance. Additionally, the surface chemistry and properties of these devices are explored via X-ray photoelectron spectroscopy, contact angle measurements, and fluorescent imaging at each reaction step, and show high density surface coverage of only the devices themselves. Based on these analyses, the optimal parameters for fabrication and surface modification protocols have been found, and result in the creation of devices whose surface functionalities have high density packing, low optical absorption, specificity only to the target ligand, and high stability in air and water, as well as high device sensitivity. Additionally, the resulting biotinylated device is used to detect streptavidin as a proof of concept.

This work represents one of the first examples non-physisorption-based bioconjugation of optical microtoroid resonators for the label-free detection of biomolecules. The primary advantage of a device that has been functionalized using the methods described above is that a wide array of biomolecules can be detected and studied, since the functionalization process does not limit the study to a specific ligand-substrate pair, or even a specific type of molecule. For example, the protocols developed here are easily extended to a wide variety of probe molecule / target molecule pairings by using the high affinity of biotin for (strept)avidin-modified probes molecules, allowing the easy interchange of the terminal functional group probes on the surface of the devices. Additionally, these methods can also be extended to a variety of silica-based optical sensors, such as microdisk and microsphere resonators, as well as waveguide-based sensors, resulting in an array of high specific sensor devices that can be utilized for rapid and real-time detection. Lastly, the optical biosensors developed during this work may be used to probe macromolecular interactions at the single-molecule level due to their ultra-high sensitivity, possibly providing insight into rare conformations, unusual reaction pathways, and fluctuating systems. This could lead to a detailed understanding of interactions that would be impossible with ensemble studies, where the behavior of individual molecules is easily disguised within ensemble averages.4

1. Armani, A. M.; Kulkarni, R. P.; Fraser, S. E.; Flagan, R. C.; Vahala, K. J. Science 2007, 317, (5839), 783-787. 2. Vollmer, F.; Arnold, S. Nature Methods 2008, 5, (7), 591-596. 3. Armani, D. K.; Kippenberg, T. J.; Spillane, S. M.; Vahala, K. J. Nature 2003, 421, (6926), 925-928. 4. Sakamoto, T.; Webb, M. R.; Forgacs, E.; White, H. D.; Sellers, J. R. Nature 2008, 455, (7209), 128-U99.