Electrochemical Detection of Multiple Cancer Protein Biomarkers using Microfluidic Device

This thesis describes the fabrication of a simple, low cost microfluidic electrochemical immunosensor for the sensitive and accurate detection of cancer protein biomarkers which holds great promise for point-of-care diagnostics. Elevated levels of proteins in blood serum that are indicative of disease states have great potential in early cancer diagnostics and therapeutic monitoring. Sensitive and accurate detection of panel of proteins with low sample consumption is necessary for accurate disease diagnostics.^ Amperometric enzyme linked immunoarrays for four cancer biomarkers, PSA, prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4) and interleukin-6 (IL-6) were developed on vertically aligned single wall nanotube (SWNT) forests on pyrolytic graphite electrodes. Sensitivity of the detection method was improved using strategy involving the use of biotinylated secondary antibodies (Ab2) that bind specifically to streptavidin-horseradish peroxidase (HRP) bioconjugates to provide 14-16 labels on each Ab2. Measurement of four biomarkers in serum of prostate cancer patients using SWNT immunoarrays showed excellent correlation with standard enzyme linked immunosorbent (ELISA) assays.^ A simple microfluidic device was fabricated featuring a microfluidic channel made from poly(dimethyl siloxane) polymer placed between two flat poly(methylmethacrylate) plates and interfaced with pump and sample injector. The device was validated by detecting hydrogen peroxide with an excellent detection limit of 5 nM using gold wire modified with glutathione-decorated gold nanoparticles linked to HRP as sensing, Ag/AgCl reference and Pt wire counter electrodes.^ The device was modified to insert 8 electrode chip and electrochemical immunoarray system was developed for the simultaneous detection of PSA and IL-6 biomarkers. Using off-line capture of analyses by enzyme-labeled 1 μm magnetic particle (MP)-antibody bioconjugates (∼90,000 antibodies and ∼200,000 HRP labels) and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of PSA and IL-6 with detection limits of 0.23 pg mL -1 and 0.30 pg mL-1 respectively was achieved in diluted serum mixture. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and assay results gave excellent correlation with standard ELISA. These microfluidic immunoarrays hold great promise for accurate, sensitive multiplexed detection of cancer biomarkers for point-of-care use.