Highly sensitive electrochemical detection of circulating EpCAM-positive tumor cells using a dual signal amplification strategy

Circulating tumor cells (CTCs) play an important role in diagnosing cancer and metastasis; thus, appropriate sensors are required for analyzing CTCs in patient samples. Here, we developed a simple electrochemical aptasensor for highly sensitive and selective detection of CTCs based on rolling circle amplification (RCA) coupled with the catalytic ability of the hemin/G-quadruplex complex. In detail, we first modified the surface of a screen-printed carbon electrode using a biocompatible mixture of thiol functionalized mesoporous silica nanoparticles and ionic liquid. Next, gold nanostar structures were prepared by electrodeposition to increase the electrode surface area and electrochemical signal, followed by EpCAM aptamer immobilization. When EpCAMpositive cancer cells are present in the sample, they bind to the EpCAM aptamer on the modified electrode, and a number of cholesterol-labeled DNA probes that act as initiators for RCA are inserted into the cell membrane. Consequently, RCA is initiated to generate multiple tandem repeats of the G-quadruplex DNA structure, which is complexed with hemin to catalyze the H2O2-mediated oxidation of 3,3',5,5'-tetramethyl benzidine and produces strong electrochemical signals. Using this strategy, we could sensitively determine target cancer cells down to a single cell with high selectivity even in human serum. The proposed strategy with the dual signal amplification can thus be a promising platform for simple and ultrasensitive detection of CTCs, and be used for the detection of other cancer cells and biomarkers in different clinical and biological samples.