Assay of Single-Cell Apoptosis by Ensemble and Single-Molecule Fluorescence Methods: Annexin-V/Polyethylene...

Apoptosis plays a critical role in many biological processes and the etiology of various diseases of the immune system. The study of apoptosis would allow both improving the diagnosis of certain diseases and serving as a target of drug screening. In this paper, we developed a sensitive assay of single-cell apoptosis using semiconductor quantum dots (QDs) as fluorescent-labeling probes. The principle of this assay is based on the detection of phosphatidylserine (PS) exposed on the plasma membrane during the drug-induced apoptosis. The QD-labeled annexin V (AV) was prepared to specifically target PS on the membrane of apoptotic cells, and PS was detected by fluorescent imaging, flow cytometer, and single-molecule fluorescence correlation spectroscopy (FCS). We developed the procedures for conjugation of QDs to AV and for purification of their conjugates by gel chromatography. The obtained conjugates were characterized by FCS, capillary electrophoresis, and zeta potential analyzer. We studied the nonspecific adsorption of cells to different surface-modified QDs and found that the nonspecific adsorption effects were significantly reduced by modification of QDs with polyethylene glycol in the detection of apoptosis. In this assay, the results obtained by flow cytometry were consistent with the commercial test kit. Furthermore, a home-built single-molecule FCS system was developed for in situ study the drug-induced apoptosis. We observed the significant change in the diffusion coefficients of QDs on cells during the progress of cell apoptosis. Compared with conventional methods, the fluorescent methods represented here possess high sensitivity because of the use of high photo stability and brightness QDs as labeling probes and provide the temp-spatial information on a single apoptotic cell.