Detection of colorectal circulating cancer cells with the use of a quantum dot labelled magnetic immunoassay method

Abstract

To evaluate the incorporation of cadmium selenide quantum dots (QDs) in the detection of colorectal cancer (CRC) circulating tumour cell (CTC) surface antigens. The principle of this assay is based upon the separation of CTCs from body fluids using magnetic beads (MBs) coupled with specific antibody biomarkers, such as the Epithelial cell Adhesion Molecule Antibody (EpCAM) and Cytokeratin 19 (CK19) antibody. The detection signal was acquired from the fluorescence signal of QDs. To evaluate the performance, the method under study was used to isolate the human colon adenocarcinoma cell line (DLD-1) and CTCs from CRC patients’ peripheral blood samples. Peripheral blood samples were obtained from 9 CRC patients (Table 1) and 5 healthy donors who gave their informed consent to their inclusion in the study. Peripheral venous blood was sampled immediately after CRC patients had received general anaesthesia, moments before surgery. In all patients, an intravenous cannula was used to collect blood into 7 ml vacutainers containing sodium ethylenediaminetetraacetic acid (EDTA). The first 7 ml of blood was discarded in order to reduce the risk of contamination from skin epithelial cells. Subsequently, 30 ml samples were collected at one-minute intervals (five 6 ml aliquots per 30 ml sample). The limit of detection (LOD) of the methodology described in the present study was determined at 10 DLD-1 cells/ml of sample as fluorescence measured with a spectrofluorometer. Fluorescence activated cell sorting (FACS) analysis and Real Time RT-PCR were also used to evaluate the performance of the method. Ultimately, in comparison to other methods currently in use, we developed a simple, sensitive, and more cost-effective method for the detection of CRC CTCs in human samples. Results were accomplished using magnetic bead isolation and subsequent QD fluorescence detection. The present method can be readily adjusted to target a variety of proteins of either the CTC or the host.