Multiplexed Detection by Whispering Gallery Mode Imaging

The term “whispering gallery” was first introduced in 1910 by Lord Rayleigh to explain the travel of sound through the dome shaped celling of St. Paul’s Cathedral in London. Today the term is also used to define the confinement of light inside a circular dielectric. These optical resonators provide an ideal platform for sensitive, robust and label-free biosensors due to their ability to detect changes in the effective refractive index of the surrounding medium. A primary objective of our research is to couple low background fluorescence imaging with microresonators to create a scalable biosensor with low detection limits. We aim to develop a multiplexed bioassay for early detection of ovarian cancer.

Ovarian cancer is one of the most prevalent gynecologic cancers and unfortunately the most fatal as it remains the fifth leading cause of cancer related deaths in North America. Of the females diagnosed with ovarian cancer, over 60% will die from it.1 This staggering statistic is in part due to the difficulty of early diagnosis; when diagnosed early, ovarian cancer is almost entirely treatable. Existing ovarian cancer diagnostic methods include pelvic ultrasounds and monitoring of the antigen CA-125. Current analytical techniques lack sensitivity and specificity preventing effective diagnosis of early stage ovarian cancer. New bioassays with improved detection limits and multiplexed detection capabilities would allow for accurate screening of ovarian cancer and aid in early diagnosis.

Whispering gallery mode imaging allows for a unique bioassay platform that provides label-free, ultrasensitive detection capabilities. The confinement of light in a microsphere results from total internal reflection (TIR) occurring at the air-dielectric interface. This confinement is achieved when a specific wavelength is coupled into the microresonator. The proper coupling wavelength needed to achieve resonance is described by the following equation:

equation

Where λ is the wavelength of light, r is the resonators radius, neff is the effective refractive index of the medium, and m is an integer. These optical resonances are known as whispering gallery modes (WGMs).

We have developed an imaging platform by fluorescently coating microresonators in order to measure their WGM via fluorescence intensity. By using WGM imaging, a multiplexed bioassay can be designed to detect multiple ovarian cancer biomarkers. To perform the assay, sensing elements for a specific biomarker, such as antigen CA-125, are attached to the resonators surface. When binding of the analyte occurs, the effective refractive index of the microresonator changes resulting in a shift of the coupling wavelength. This provides a detectable sensor signal to quantitatively measure the analyte present in the biological sample. To complete the assay, a TIR microscope is used for excitation of the microsphere WGM sensor. Fluorescence from the WGM is collected and imaged from above using an upright microscope.

WGM1 image
Figure 1. Whispering gallery mode imaging set up with TIRM exctition and upright microscope collection.

Sensor multiplexed capabilities are achieved by using microspheres of different sizes. Each sphere size is encoded with a specific sensing element to differentiate sensor specificity.

WGM2 Image
Figure 2. Multiplexed assay design with specific sensing elements attached to microspheres of various sizes. When excited, a WGM shift in a sphere of a specific size indicates the presence of a particular biomarker.

Our results indicate the feasibility of this method to simultaneously detect two ovarian cancer biomarkers- tumor necrosis factor-alpha (TNF-α) and CA-125. A completed bioassay using 38 µm diameter anti-CA-125 labeled spheres showed a linear response to the addition of CA-125 with an R2 value of 0.9677 and a determined LOD of   ̴ 0.75 U/mL;  an improvement from traditional assay methods such as ELISA. Future directions of the project include investigating the mode patterns of the whispering galleries as well as modeling the phenomena with COMSOL. We are currently investigating changes in the mode structure as a possible mechanism for qualitative and quantitative sample analysis.

WGM3 Image

WGM4 Image
Figure 3. Whispering gallery mode images of fluorescently labeled sphere excited at different wavelengths (Top). Images generated from COMSOL modeling of WGM phenomena (Bottom).

References

  1. Chambers, A. F.; Vanderhyden, B. C., Ovarian cancer biomarkers in urine. Clin. Cancer Res. 2006, 12 (Copyright (C) 2011 American Chemical Society (ACS). All Rights Reserved.), 323-327

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