Precise Capture and Direct Quantification of Tumor Exosomes via a Highly Efficient Dual-Aptamer Recognition-Assisted Ratiometric Immobilization-Free Electrochemical Strategy

Tumor exosomes are promising biomarkers for early cancer diagnosis in a noninvasive manner. However, precise capture and direct analysis of tumor-specific exosomes in complex biological samples are still challenging. Herein, we present a highly efficient dual-aptamer recognition system for precisely isolating and quantifying tumor exosomes from the complex biological environment based on hyperbranched DNA superstructure-facilitated signal amplification and ratiometric dual-signal strategies. When tumor exosomes were captured by the dual-aptamer recognition system, the cholesterol-modified DNA probe was anchored on the surface of the exosomes, activating DNA tetrahedron-based hyperbranched hybridization chain reaction to generate a sandwich complex. Then, the sandwich complex could bind a large number of Ru(NH₃)₆³⁺ (Ru(III)), leading to a small amount of unbound Ru(III) left in the supernatant after magnetic separation. Hence, the redox reaction between Ru(II) and [Fe(CN)₆]^3- (Fe(III)) was significantly prevented, causing an obviously enhanced I_Fe(III)/I_Ru(III) value. Consequently, highly sensitive detection of tumor exosomes was achieved. The developed approach successfully realized direct isolation and analysis of tumor exosomes in complex sample media and human serum samples as well. More significantly, this ratiometric dual-signal mode and immobilization-free strategy effectively circumvented the systematic errors caused by external factors and the tedious probe immobilization processes, thus displaying the excellent performances of high reliability, improved accuracy, and easy manipulation. Overall, this approach is expected to offer novel ways for nondestructive early cancer diagnosis.