Quantum dot-based microfluidic immunocytochemistry using a parallel-sequential multiplexing method

Abstract

Immunocytochemistry (ICC) has been widely used for the cell study, such as cell to cell interaction, signal pathway and protein translocation. Particularly, multiplexed protein identification is an important issue on ICC field to explain the complicated cell signaling behavior. Conventional quantum dot (QD)-based ICC has been used to satisfy the need for multiplexing biomarkers. QD multiplexing methods are classified into two methods which are the direct method and the indirect method. Although the indirect method is preferred in QD-ICC due to its brightness and flexibility, it has limitations on multiplexing efficiency and the non-specific binding of QD-secondary antibody. In this thesis, we propose a QD parallel-sequential multiplexing method which provides high multiplexing efficiency to QD indirect method. To demonstrate this novel method, a multichannel-microfluidic device was exploited to investigate the result distortion of QD staining data resulted from the sequence dependency of QD multiplexing. With a precise control of fluid flow in the microfluidic device, we successfully showed reduced non-specific binding of QDs comparing to conventional method. We also demonstrated the feasibility of a QD parallel-sequential method with five different primary antibodies and two kinds of QDs. This parallel-sequential method could realize the massive multiplexed protein identification with reducing time, reagents, costs and labors. Reduced non-specific binding of QDs with this approach could offer the realization of massive protein quantification on ICC.