PROGRESS IN DEVELOPING AN OVARIAN CANCER SCREEN BASED ON WHISPERING GALLERY MODE IMAGING

Despite considerable research devoted to identifying biomarkers for ovarian cancer, no screening method is currently available for routine early detection of the disease. While highly treatable in its early stages, a lack of symptoms early in the disease progression often results in the cancer spreading to other tissues before being diagnosed, significantly reducing the survival rate. CA-125 is currently the most common biomarker used for the detection of ovarian cancer; however its poor diagnostic reliability when measured alone necessitates the use of a multiplexed biomarker screen for accurate early-stage diagnosis. Numerous protein biomarkers have been identified for this purpose as well as small, noncoding oligonucleotides referred to as microRNAs. New assays for screening this combination of multiple analyte types require the capability of detecting any class of target, including proteins and nucleic acids. This work describes progress in developing a label-free, multiplexed assay for the early detection of ovarian cancer. The instrumental approach exploits tiny microresonators that take advantage of a phenomenon referred to as whispering gallery mode (WGM) resonances. This method is based on the ability of circular dielectrics to store light through continuous total internal reflection, where the wavelengths stored are dependent upon the surrounding refractive index. By attaching capture agents to the sensor surface, binding events change the local RI and shift the resonant wavelength which can be used to quantify analyte binding. To combine the favorable attributes of two traditional WGM techniques, in Chapter 2 we introduce a new approach for biosensing which combines microsphere WGM resonators with fluorescence imaging to create a flexible platform for multiplexed biosensing. It is shown that the multiplexing capability is vastly increased using this imaging technique as compared to other WGM methods, enabling the detection of multiple targets in a single assay. The feasibility of using this method for biomarker measurement is demonstrated by detecting two biomarkers linked to ovarian cancer pathogenesis, CA-125 and tumor necrosis factor-á. To advance the WGM imaging technique toward clinical use, we show in Chapter 3 that non-specific binding is not limiting by detecting CA-125 doped into serum samples. The eventual clinical utility of this approach is illustrated by measuring CA-125 levels in serum collected from a healthy donor and a patient diagnosed with ovarian cancer. Although whispering gallery detection is effective in biomarker measurement, in practice the sensing region covers a very small area. As in all label-free assays, non-specific binding can be an issue for WGM detection and it is desirable that all immobilized antibodies be active. It is crucial, therefore, that all capture antibodies are immobilized in the appropriate orientation for efficient analyte binding. To maximize sensor performance, therefore, new techniques capable of characterizing antibody orientation at the single molecule level are required. A new approach for measuring single antibody orientations is therefore introduced and validated in well-characterized test systems using model lipid membranes. It has been shown that polarized total internal reflection fluorescence microscopy (PTIRF-M) is capable of directly determining the three-dimensional orientation of single molecules. Using Langmuir-Blodgett monolayers of DPPC, we show in Chapter 5 that these orientations can be used to track membrane structure at the molecular level. We utilize this to find the characteristic "equivalent surface pressure" between lipid monolayers formed by Langmuir-Blodgett deposition and supported lipid bilayers formed by vesicle fusion. The single molecule approach is further explored by comparing the condensing effects of sterol additives in each system. These measurements show that orientations respond to the addition of condensing sterols, further supporting the notion that this technique can track structural changes at the molecular level. As future single-molecule work will be used to determine antibody orientations in a dynamic, fluid environment, it is important to explore the time-resolved capabilities of this approach. In Chapter 6 we show that single molecule orientational changes can be captured with ~1s temporal resolution. By increasing the hydration levels of model membrane systems through changes in relative humidity, we show that both structural and dynamic changes occur within the membrane at the molecular level. In addition, it was observed that this technique is capable of measuring three-dimensional orientational changes in real time. Future goals include employing this method to probe antibody orientations on substrates in situ, so these studies are an important step for understanding how the technique may be applied in fluidic systems

Single molecule probes of membrane structure: Orientation of BODIPY probes in DPPC as a function of probe structure

Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir–Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe