Development of a quantum-dot-labelled magnetic immunoassay method for circulating colorectal cancer cell detection

Abstract

Aim: To detect of colorectal cancer (CRC) circulating tumour cells (CTCs) surface antigens, we present an assay incorporating cadmium selenide quantum dots (QDs) in these paper.

Methods: The principle of the assay is the immunomagnetic separation of CTCs from body fluids in conjunction with QDs, using specific antibody biomarkers: epithelial cell adhesion molecule antibody, and monoclonal cytokeratin 19 antibody. The detection signal was acquired from the fluorescence signal of QDs. For the evaluation of 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.

Results: The minimum detection limit of the assay was defined to 10 DLD-1 CRC cells/mL as fluorescence was measured with a spectrofluorometer. Fluorescence-activated cell sorting analysis and Real Time RT-PCR, they both have also been used to evaluate the performance of the described method. In conclusion, we developed a simple, sensitive, efficient and of lower cost (than the existing ones) method for the detection of CRC CTCs in human samples. We have accomplished these results by using magnetic bead isolation and subsequent QD fluorescence detection.

Conclusion: The method described here can be easily adjusted for any other protein target of either the CTC or the host.

Keywords: Cancer; Circulating tumor cells; Micrometastasis; Nanoprobes; Quantum dots.

Figure 1

Circulating tumour cells are separated from the leucocytes using magnetic beads coupled with epithelial cell adhesion molecule antibody and a magnetic device. These complexes are then tagged with cytokeratin-19 (CK19) biotinylated antibody and streptavidin-conjugated quantum dots (QDs) which lead to the detection of a fluorescent signal. EpCAM: Epithelial cell adhesion molecule; DLD-1: Human colon adenocarcinoma cell line.

Figure 2

Representative fluorescence-activated cell sorting analysis of peripheral blood samples. A: Events which fell within region R1 are confirmed to be cytokeratin 19 (CK19)+ and fluorescent in FL3 (anti-CK19 conjugated with QDs); B: From the R1 gate, the events confirmed to be CK19+CD45-are those in the UL region, in UR region the events represent those that are CK19+CD45+ and fluorescent in FL1 (anti-CD45 conjugated with Alexa Fluor). We deemed that the cells in the UL are those meeting the criteria for circulating tumor cells.

Figure 3

Representative results recorded by the proposed method for the human colon adenocarcinoma cell line serial dilutions ranging from 10⁴ to 10 cells/mL (numbers 1-5) and negative controls. Fluorescence is evident in the human colon adenocarcinoma cell line samples with concentration up to 10 cells/mL, as illustrated at the fluorescent emission spectra obtained after magnetic separation.