MP14:Chennapragada:Nanochannel Immunoassay sensor for Cardiac protein biomarker detection
Pavani Chennapragada1, Krishna Vattipalli1, Thomas Barrett2 and Shalini Prasad1,*
1Department of Bioengineering, University of Texas at Dallas, Richardson, TX – 75080
2Oregon Health Sciences University, Portland, OR - 97239
Troponin T (TNT) is a diagnostic cardiac biomarker. It modulates the contraction of striated muscle. This biomarker is released into the blood when the heart is damaged. Measurements of this biomarker will help us identify the presence of acute coronary syndrome in a patient. Current technology by Roche, Elecsys Troponin-T high sensitive assay has a detection limit of 5 pg/mL. This immunoassay was based on electrochemi-luminescence. We have devised a miniaturized microarray platform which leverages the principles of nanoconfinement to enhance the limit of detection as well as the linear dynamic range of detection. We have demonstrated sensitivity at 0.1 fg/mL with linear dynamic range of 0.1fg/mL to 10 ng/mL to detect Troponin-T. A standard “single-capture” enzyme-linked immunosorbent assay (ELISA)–style immunoassay (linker-capture antibody–target antigen assay) was designed and incorporated onto alumina nanochannels, fabricated through electrochemical anodization and integration with standard top-down lithography. The generated alumina nanowells have gold base electrodes and a large number of nanowells can be connected such that signal amplification may be achieved during detection. The key idea is the miniaturization of the microarray technology so as to enhance the protein association. We hypothesize that due to the planar nature of the multi wells of a protein microarray platform, the association of the protein with the antibody does not occur, especially at low protein concentrations due to the loss of efficacy of the proteins as well as due to the significantly larger volume within each well in comparison to the size of the protein. The goal of the designed nanowell diagnostic platform is to detect troponin-T at the femtogram/mL sensitivity with a detection speed in the order of minutes, which is critical for designing rapid care diagnostics tools. The biosensor design mimics protein confinement within biological systems. The macromolecular crowding theory in cellular biophysics postulates that there exists a crowded environment inside the cell namely the cytoplasm which is typically different from the dilute solutions that are generally used in the in-vitro studies of proteins. The protein binding and detection process is achieved by incorporating an immunoassay into each nanowell of the biosensor. All the biomolecules have surface charges. The binding of these molecules to the base of each nanowell perturbs the charge distribution in the electrical double layer that forms at the solid/liquid interface. This charge perturbation produces a capacitance change in the electrical double layer. The biomolecule binding induced capacitance change can be measured by the electrical impedance spectroscopy technique. This technique is widely used in electrical biosensors for detecting surface charged analytes.
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