Abstract 4067: Integrated microfluidic bead-based immunosensor for multiplexed determinations of ovarian cancer biomarkers at the point-of-care
Authors: Raamanathan Archana , Shadfan Basil , Simmons Glennon W. , Ho Andy , Bast Robert C. , McDevitt John 
Published: August 2012

Abstract
Early detection of ovarian cancer could significantly impact clinical outcome and hence patient survival. Given the biological heterogeneity of ovarian cancer, multi-marker panels will be required. Validation of such panels must utilize pre-clinical serum samples that are available only in small quantities. Diagnostic platforms that are capable of rapid, precise and multiplexed analysis of biomarkers in small quantities of serum should facilitate validation of optimal biomarker panels. Once validated, these platforms could permit rapid screening with finger-stick quantities of blood at point-of-care. The Programmable Bio-Nano-Chip (p-BNC) microfluidic immunosensor provides such a platform. In the p-BNC, serum biomarkers are sequestered and assessed with a fluorescence-based sandwich immunoassay, completed in nano-nets of sensitized agarose microbeads localized in individually addressable wells, housed in a microfluidic module, capable of integrating multiple sample, reagent and biowaste processing and handling steps. Previously, we adapted the p-BNC for the quantification of the CA125 biomarker with low LODs, high precision and a short analysis time of 43 minutes. Here, we adapt the p-BNC to simultaneously assess CA125 and HE4, a promising biomarker panel implicated in early detection. The HE4 immunoassay was developed to perform optimally with the previously established CA125 assay as the rate determining step. Extensive cross reactivity analysis ruled out non-ideal interactions between HE4 and CA125 reagents at the level of capturing antibody, detecting antibody and individual analytes. Wash times were optimized to maximize p-BNC performance. The dose-response curves obtained for the individual analytes (singleplex) were similar to those obtained with multiplex assays. Identical LOD values were noted for the singleplex and multiplex immunoassays with 1.5-fold decrease in precision for the multiplexed assays, demonstrating negligible loss of analytical performance upon multiplexing. For clinical validation, 8 surgically confirmed advanced stage patient sera and 8 age-matched healthy controls were assessed on the multiplexed p-BNC. ROC curve analysis for the CA125-HE4 biomarker combination, interpreted with a Risk of Ovarian Malignancy Algorithm was able to discriminate diseased samples from healthy controls with an AUC of 1.00. The p-BNC indicates strong promise for robust, multiplexed, quantitative measurements of ovarian cancer biomarkers, with high precision and sensitivity to yield clinically pertinent information under 45 minutes. Incorporation of additional biomarkers is underway to establish and validate an optimal panel for early detection of ovarian cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4067. doi:1538-7445.AM2012-4067