Rapid Detection of Avian Mitochondrial Proteins Using Capillary Column Immunosensor

Abstract

Proteomics is increasingly used to gain greater understanding of the many functions of mitochondria in metabolism and metabolic diseases. Immunochemical detection has high specificity and sensitivity, and large sample throughput. The use of antibody/antigen-modified capillary columns presents advantages over traditional immunochemical methods by improving assay kinetics due to higher surface-to-volume ratio and restricted diffusion of compounds. Capillary columns are more suitable for automation, and consumption of immunoreagents is minimized due to the small dimensions of capillaries. With the realization of significance of proteins in metabolism and metabolic diseases, a high-throughput instrument for the detection of proteins is of increasing importance. A capillary column immunosensor was developed for rapid detection of proteins in chicken lymphocyte samples based on their optical characteristics. For detection of proteins, capillary columns were modified with a primary antibody. After capture of proteins, a secondary antibody was applied and detected using enzyme labeled anti-IgG and substrate. For vinculin, the linear detection range was 25~-200 ng/mL (0.22-1.72 nM), assay precision was 3~23%, and the lower limit of detection (LOD) was calculated to be 4.6 ng/mL (40 pM) at a signal-to-noise ratio (S/N) of 3. For Cyt c, a linear detection range of 10-50 ng/mL (0.83-4.2 nM) was achieved with an LOD of 5.1 ng/mL (0.42 nM) and a precision of 8~27%. The biosensor was more sensitive than ELISA by one order of magnitude in terms of LOD and sensitivity was suitable for detection of target proteins in broiler lymphocytes. Detection time was 1.5-h and multiple samples were analyzed simultaneously.