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A Single-Molecule Homogeneous Immunoassay by Counting Spatially “Overlapping” Two-Color Quantum Dots with Wide-Field Fluorescence Microscopy
journal contribution
posted on 2018-11-14, 00:00 authored by Xiaojun Liu, Conghui Huang, Chenghua Zong, Aiye Liang, Zhangjian Wu, Yusu Zhang, Qingquan Zhang, Wenfeng Zhao, Hongwei GaiWe developed a single-molecule homogeneous
immunoassay by counting
spatially “overlapping” two-color quantum dots (QD)
under a wide-field fluorescence microscope. QD 655 with red fluorescence
and QD 565 with green fluorescence were modified with capture and
detection antibodies, respectively. A capture antibody-modified QD
655 and a detection antibody-modified QD 565 were conjugated by a
corresponding antigen molecule to form a “sandwich”
immunocomplex. The conjugated QD 655 could not be distinguished from
the conjugated QD 565 by fluorescent microscopy because the distance
between them was smaller than the resolution of an optical microscope
(approximately 200 nm). The immunocomplex color became yellow because
of the spatial “overlap” of the red and green fluorescence.
The number of the yellow spots was equal to the number of immunocomplex
molecules, while the concentration of the antigen was related to the
ratio of the yellow dots to the red dots. The successful quantification
of two model proteins in the human plasma, namely, alpha-fetoprotein
and carcinoembryonic antigen, demonstrated the accuracy and reliability
of our approach.