Heather K. Hunt

Bioconjugation Strategies for Microtoroidal Optical Resonators

The development of label-free biosensors with high sensitivity and specificity is of significant interest for medical diagnostics and environmental monitoring, where rapid and real-time detection of antigens, bacteria, viruses, etc., is necessary. Optical resonant devices, which have very high sensitivity resulting from their low optical loss, are uniquely suited to sensing applications. However, previous research efforts in this area have focused on the development of the sensor itself. While device sensitivity is an important feature of a sensor, specificity is an equally, if not more, important performance parameter. Therefore, it is crucial to develop a covalent surface functionalization process, which also maintains the device’s sensing capabilities or optical qualities. Here, we demonstrate a facile method to impart specificity to optical microcavities, without adversely impacting their optical performance. In this approach, we selectively functionalize the surface of the silica microtoroids with biotin, using amine-terminated silane coupling agents as linkers. The surface chemistry of these devices is demonstrated using X-ray photoelectron spectroscopy, and fluorescent and optical microscopy. The quality factors of the surface functionalized devices are also characterized to determine the impact of the chemistry methods on the device sensitivity. The resulting devices show uniform surface coverage, with no microstructural damage. This work represents one of the first examples of non-physisorption-based bioconjugation of microtoroidal optical resonators.

Determination of binding kinetics using whispering gallery mode microcavities

Silica optical microcavity sensors show great promise in the kinetic evaluation of binding pairs, fundamental in understanding biomolecular interactions. Here, we develop and demonstrate a novel platform, based on bioconjugated silica microsphere resonators, to study the binding kinetics of the biotin-streptavidin system. We characterize the optical performance, verify the covalent attachment of biotin to the surface, and perform streptavidin detection experiments. We perform preliminary kinetic analysis of the detection data which shows the potential of whispering gallery mode resonators in the determination of the dissociation constant of the binding pair, which is in good agreement with previously published values.

Bioconjugation Strategies for Label-Free Optical Microcavity Sensors

Whispering gallery mode optical microcavities have significantly impacted the field of label-free optical biodetection. By combining the evanescent field generated by the microcavity with biomimetic surface chemistries, it is now possible to use the microcavities as not only biosensors, but as analytical tools to explore fundamental chemical and physical interactions of biomolecules and biomaterials. Here, we review the recent advancements of these applications from a surface chemistry perspective. For example, surface chemistries can be generated from a standard coating perspective, where active molecules, such as laser or fluorescent dyes can be embedded in a biomaterial matrix. Alternatively, direct and reverse grafting techniques can be used to tether biomolecules of interest to the surface to tune the surface properties (hydrophobicity/hydrophilicity, protein adsorption, cell adhesion, etc.). Finally, we discuss how to apply advancements in biomimetic chemistry from other sensor approaches to these devices to continue the development of new analytical tools. All of these developments rely on a firm understanding of how proper surface chemistries can be merged with whispering gallery mode optical microcavities to achieve not just a platform, but a precisely defined tool for a given application.

Recycling microcavity optical biosensors

Optical biosensors have tremendous potential for commercial applications in medical diagnostics, environmental monitoring, and food safety evaluation. In these applications, sensor reuse is desirable to reduce costs. To achieve this, harsh, wet chemistry treatments are required to remove surface chemistry from the sensor, typically resulting in reduced sensor performance and increased noise due to recognition moiety and optical transducer degradation. In the present work, we suggest an alternative, dry-chemistry method, based on O2 plasma treatment. This approach is compatible with typical fabrication of substrate-based optical transducers. This treatment completely removes the recognition moiety, allowing the transducer surface to be refreshed with new recognition elements and thus enabling the sensor to be recycled.

PEG Functionalization of Whispering Gallery Mode Optical Microresonator Biosensors to Minimize Non-Specific Adsorption during Targeted, Label-Free Sensing.

Whispering Gallery Mode (WGM) optical microresonator biosensors are a powerful tool for targeted detection of analytes at extremely low concentrations. However, in complex environments, non-specific adsorption can significantly reduce their signal to noise ratio, limiting their accuracy. To overcome this, poly(ethylene glycol) (PEG) can be employed in conjunction with appropriate recognition elements to create a nonfouling surface capable of detecting targeted analytes. This paper investigates a general route for the addition of nonfouling elements to WGM optical biosensors to reduce non-specific adsorption, while also retaining high sensitivity. We use the avidin-biotin analyte-recognition element system, in conjunction with PEG nonfouling elements, as a proof-of-concept, and explore the extent of non-specific adsorption of lysozyme and fibrinogen at multiple concentrations, as well as the ability to detect avidin in a concentration-dependent fashion. Ellipsometry, contact angle measurement, fluorescence microscopy, and optical resonator characterization methods were used to study non-specific adsorption, the quality of the functionalized surface, and the biosensor’s performance. Using a recognition element ratio to nonfouling element ratio of 1:1, we showed that non-specific adsorption could be significantly reduced over the controls, and that high sensitivity could be maintained. Due to the frequent use of biotin-avidin-biotin sandwich complexes in functionalizing sensor surfaces with biotin-labeled recognition elements, this chemistry could provide a common basis for creating a non-fouling surface capable of targeted detection. This should improve the ability of WGM optical biosensors to operate in complex environments, extending their application towards real-world detection.

Application of bacteriophages to selectively remove Pseudomonas aeruginosa in water and wastewater filtration systems

Water and wastewater filtration systems often house pathogenic bacteria, which must be removed to ensure clean, safe water. Here, we determine the persistence of the model bacterium Pseudomonas aeruginosa in two types of filtration systems, and use P. aeruginosa bacteriophages to determine their ability to selectively remove P. aeruginosa. These systems used beds of either anthracite or granular activated carbon (GAC), which were operated at an empty bed contact time (EBCT) of 45 min. The clean bed filtration systems were loaded with an instantaneous dose of P. aeruginosa at a total cell number of 2.3 (± 0.1 [standard deviation]) × 10(7) cells. An immediate dose of P. aeruginosa phages (1 mL of phage stock at the concentration of 2.7 × 10(7) PFU (Plaque Forming Units)/mL) resulted in a reduction of 50% (± 9%) and >99.9% in the effluent P. aeruginosa concentrations in the clean anthracite and GAC filters, respectively. To further evaluate the effects of P. aeruginosa phages, synthetic stormwater was run through anthracite and GAC biofilters where mixed-culture biofilms were present. Eighty five days after an instantaneous dose of P. aeruginosa (2.3 × 10(7) cells per filter) on day 1, 7.5 (± 2.8) × 10(7) and 1.1 (± 0.5) × 10(7) P. aeruginosa cells/g filter media were detected in the top layer (close to the influent port) of the anthracite and GAC biofilters, respectively, demonstrating the growth and persistence of pathogenic bacteria in the biofilters. A subsequent 1-h dose of phages, at the concentration of 5.1 × 10(6) PFU/mL and flow rate of 1.6 mL/min, removed the P. aeruginosa inside the GAC biofilters and the anthracite biofilters by 70% (± 5%) and 56% (± 1%), respectively, with no P. aeruginosa detected in the effluent, while not affecting ammonia oxidation or the ammonia-oxidizing bacterial community inside the biofilters. These results suggest that phage treatment can selectively remove pathogenic bacteria with minimal impact on beneficial organisms from attached growth systems for effluent quality improvement.

The Detection of Helicobacter hepaticus Using Whispering-Gallery Mode Microcavity Optical Sensors

Current bacterial detection techniques are relatively slow, require bulky instrumentation, and usually require some form of specialized training. The gold standard for bacterial detection is culture testing, which can take several days to receive a viable result. Therefore, simpler detection techniques that are both fast and sensitive could greatly improve bacterial detection and identification. Here, we present a new method for the detection of the bacteria Helicobacter hepaticus using whispering-gallery mode (WGM) optical microcavity-based sensors. Due to minimal reflection losses and low material adsorption, WGM-based sensors have ultra-high quality factors, resulting in high-sensitivity sensor devices. In this study, we have shown that bacteria can be non-specifically detected using WGM optical microcavity-based sensors. The minimum detection for the device was 1 × 104 cells/mL, and the minimum time of detection was found to be 750 s. Given that a cell density as low as 1 × 103 cells/mL for Helicobacter hepaticus can cause infection, the limit of detection shown here would be useful for most levels where Helicobacter hepaticus is biologically relevant. This study suggests a new approach for H. hepaticus detection using label-free optical sensors that is faster than, and potentially as sensitive as, standard techniques.

Selective Patterning of Si-based Biosensor Surfaces Using Isotropic Silicon Etchants

Ultra-sensitive, label-free biosensors have the potential to have a tremendous impact on fields like medical diagnostics. For the majority of these Si-based integrated devices, it is necessary to functionalize the surface with a targeting ligand in order to perform specific biodetection. To do this, silane coupling agents are commonly used to immobilize the targeting ligand. However, this method typically results in the bioconjugation of the entire device surface, which is undesirable. To compensate for this effect, researchers have developed complex blocking strategies that result in selective patterning of the sensor surface. Recently, silane coupling agents were used to attach biomolecules to the surface of silica toroidal biosensors integrated on a silicon wafer. Interestingly, only the silica biosensor surface was conjugated. Here, we hypothesize why this selective patterning occurred. Specifically, the silicon etchant (xenon difluoride), which is used in the fabrication of the biosensor, appears to reduce the efficiency of the silane coupling attachment to the underlying silicon wafer. These results will enable future researchers to more easily control the bioconjugation of their sensor surfaces, thus improving biosensor device performance.

Interfacing whispering gallery mode optical microresonator biosensors with the plant defense elicitor chitin.

The biomaterial class of chitooligosaccharides (chitin), commonly found in insects and fungi, is one of the most abundant on earth. Substantial evidence implicates chitin in mediating a diverse array of plant cellular signaling events, including the induction of plant defense mechanisms against invading pests. However, these recognition and mediation mechanisms, including the binding kinetics between chitin and their plant recognition receptors, are not fully understood. Therefore, the creation of a platform capable of both interfacing with chitin and plant cell receptors, and monitoring their interactions, would significantly advance our understanding of this plant defense elicitor. Recently, a label-free, highly sensitive biosensor platform, based on Whispering Gallery Mode optical microresonators, has been developed to study such biomolecular interactions. Here, we demonstrate how this unique platform can be interfaced with chitin using simple carbohydrate chemistry. The surface chemistry is demonstrated using X-ray photoelectron spectroscopy, fluorescence microscopy, optical profilometry, ellipsometry, and contact angle measurements. The resulting surface is uniform, with an average surface roughness of 1.25nm, and is active toward chitin recognition elements. Optical loss measurements using standard quantitative cavity analysis techniques demonstrate that the bioconjugated platforms maintain the high performance (Q>10(6)) required to track binding interactions in this system. The platform is able to detect lectin, which binds COs, at 10μg/mL concentration. This biosensor platforms unique capabilities for label-free, high sensitivity biodetection, when properly interfaced with the biomaterials of interest, could provide the basis for a robust analytical technique to probe the binding dynamics of chitin-plant cell receptors.

Photoacoustic measurement of refractive index of dye solutions and myoglobin for biosensing applications

Current methods of determining the refractive index of chemicals and materials, such as ellipsometry and reflectometry, are limited by their inability to analyze highly absorbing or highly transparent materials, as well as the required prior knowledge of the sample thickness and estimated refractive index. Here, we present a method of determining the refractive index of solutions using the photoacoustic effect. We show that a photoacoustic refractometer can analyze highly absorbing dye samples to within 0.006 refractive index units of a handheld optical refractometer. Further, we use myoglobin, an early non-invasive biomarker for malignant hyperthermia, as a proof of concept that this technique is applicable for use as a medical diagnostic. Comparison of the speed, cost, simplicity, and accuracy of the techniques shows that this photoacoustic method is well-suited for optically complex systems.