Electrochemiluminescence-Based Detection of Cell Surface Biomolecules By Using Cup-Shaped Microelectrodes

Abstract

Identification of biomolecules expressed on a cell surface is important, because their expressions usually imply condition of the cell, and fluorescence-based labeling and detection is one popular method for the identification of such molecules. In the method, specific antibodies are conjugated with fluorescent dyes, target molecules are reacted with the fluorescent antibodies, and expressions of the target molecules are identified with fluorescence observation. This method is target-specific and highly sensitive, however, detection sensitivity of the method is sometimes still insufficient. For example, one problem for the identification of circulating tumor cells (CTCs) is false-negative results caused on the missing of cancer cells on which target marker molecules are few expressed. Using of electrochemiluminescence (ECL)-based detection is one useful way to improve the sensitivity [1], however, ECL can be only irradiated from very close position of the electrode, and detection of cell surface molecules by ECL-based method is difficult because of large size of cells. We propose a new method to detect cell surface biomolecules with ECL-based method by using cup-shaped microelectrodes. The hemispherical electrodes were composed of thin double layer [2], and in detail, the inner concave was low noise nano-carbon layer [3] and the outer was nickel. Diameter of the cup was almost the same as that of general animal cells [4], therefore, large area of the cell can be closely approached to the electrode surface with capture of the cell to inner concave of the cup. Epithelial cell adhesion molecule (EpCAM), which is a famous marker molecule for the identification of CTCs, was used for a model target in this study, and a human breast cancer cell line on which EpCAM was low expressed (MDA-MB-231) was used for the evaluation of EpCAM detection by ECL-based measurement. ECL probe (ruthenium(II) tris(bipyridine))-conjugated anti-EpCAM antibody was reacted with the cancer cells, the labeled cells were captured to inner concave of the cup-shaped microelectrodes, and ECL observation was performed. When electric voltage was applied, ECL was observed from cups in which cancer cells were captured. These results indicate that ECL-based detection of cell surface molecules can be achieved by using cup-shaped microelectrodes with quite high detection sensitivity.