Highly Sensitive Method for Assay of Drug-Induced Apoptosis Using Fluorescence Correlation Spectroscopy

Abstract

Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs.

Analysis of the concentrations and size distributions of cell-free DNA in schizophrenia using fluorescence correlation spectroscopy

“…Fluorescence correlation spectroscopy (FCS) is a technique for single-molecule detection that analyzes the fluctuations in fluorescence resulting from the Brownian motion of fluorescent molecules in a very small detection volume 19–21 . Previous study using FCS has demonstrated DNA molecules of different sizes released from cells following drug-induced apoptosis 22 . As noted in our previous study, a maximum entropy method (MEM)-based fitting procedure to analyze FCS raw data, called MEMFCS 23 , was successfully used to investigate the size distributions of DNA molecules in the multiple-component model.…”

Size Distribution of Nanoparticles in Solution Characterized by Combining Resonance Light Scattering Correlation Spectroscopy with the Maximum Entropy Method

“… Maiti et al first applied MEM to FCS and developed the MEMFCS method . MEMFCS has been successfully applied to study the diffusion of polymer chains, Alzheimer’s disease, drug-induced apoptosis, etc. − In the theoretical part of our work, a theoretical diffusion model of RLSCS was proposed by introducing the Mie scattering theory. This model is derived from FCS, but its key part has been revised due to some intrinsic characteristics of noble metal nanoparticles.…”

Sensitive and homogenous immunoassay of fumonisin in foods using single molecule fluorescence correlation spectroscopy

“…34 So far, FCS has been used in immunoassays, 35 drug screening, 36 and detection of drug-induced cell apoptosis. 37 In this paper, we reported a homogeneous immunoassay for the sensitive detection of FB 1 in maize. In this study, a competitive immunoassay model was used, and uorescent labeled FB 1 was used as a tracer.…”