Ultrasensitive Arrays for Multiplexed Detection of Oral Cancer Serum Biomarker Proteins in the Clinic

The occurrence of cancer involves altered protein expression, known as biomarkers of disease. The serum levels of biomarker proteins are used as indicators to differentiate between diseased and healthy states, and to monitor disease progression. Sensitive measurement of proteins over-expressed in individuals with cancer holds excellent promise for early disease detection and personalized therapies. However, the required point-of-care measurement has yet to be broadly realized. Thus, there is an urgent need for simpler, faster and inexpensive detection of serum biomarkers with high sensitivity and accuracy. In this dissertation, new methodologies for ultrasensitive multiplexed detection of serum biomarker proteins for oral cancer, as well as other malignancies, utilizing different aspects of nanotechnology in conjunction with electrochemical detection have been developed and characterized. The first part of the approach addresses the study of interleukin-6 (IL-6) using single-walled carbon nanotube forests based electrochemical immunosensor on pyrolytic graphite (PG) electrodes. With two levels of multi-enzyme labeling in a sandwich immunoassay format, 25 fM human IL-6 in serum, and experimental samples, was reproducibly measured. ^ The second part features 8-sensor microfluidic array equipped with four specific capture antibodies to detect ultralow levels of interleukin-6 (IL-6), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor C (VEGF-C) proteins, and these proteins when captured off-line using magnetic beads derivatized with 120,000 antibodies and 400,000 enzyme labels, greatly decreased non-specific binding and enhanced sensitivity. This approach provided unprecedented detection limits in low fg mL−1 range for all four biomarkers in calf serum, essentially used as a human serum surrogate. This oral cancer biomarker panel was validated by detecting proteins in sera of oral cancer patients and cancer-free controls. Receiver operating characteristic (ROC) analysis showed that a normalized combination of the levels of all 4 biomarkers enhanced the clinical discrimination of oral cancer from non-cancer conditions compared to single biomarkers, with 89% sensitivity and 98% specificity. ^ The use of microfluidic immunoarrays for multiplexed detection of biomarker proteins offers distinct advantages. The capability and versatility of these methodologies for simultaneous, sensitive, accurate, low cost real-time monitoring of protein biomarkers with high clinical selectivity and sensitivity holds excellent promise for diagnosis and prognosis of cancer with potential for rationalizing treatment options. ^ Also described in this dissertation is the development of electrochemical sensors for peanut allergen Ara h2 in serum, along with various analytical techniques to characterize the sensors to establish the primary reasons for high sensitivity and selectivity.