Ultra-Sensitive Colorimetric Assay System Based on the Hybridization Chain Reaction-Triggered Enzyme Cascade Amplification

A versatile and ultrasensitive colorimetric detection platform has been developed based on the hybridization chain reaction (HCR)-triggered enzyme cascade amplification in this work. The proposal involves the preparation of two different hairpin DNA strands consisting of the H1, modified with glucose oxidase (GOx-H1) and H2, modified with horseradish peroxidase (HRP-H2). The H1 and H2 were composed of complementary sequence of nucleic acid target (T) and interlaced complementary stem-loop sequences. In the nucleic acid detection, the hybridization of T and its complementary sequence induces the autonomous assembly of GOx-H1 and HRP-H2 through the predictable HCR, accompanied by the formation of GOx/HRP enzyme pairs with a multiple enzymatic cascade. In contrast to the crude mixture of free GOx-H1 and HRP-H2, the catalytic performance of enzyme cascade reaction has been significantly enhanced, which can be determined by monitoring the absorbance change of 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS2–), a typical substrate with hydrogen peroxide for the HRP. Furthermore, this platform can be utilized in the assay of biological substances by the introduction of corresponding aptamer (Apt), complementary strands (Com), and an assistant hairpin DNA strand (HAssist). In view of the signal amplification of HCR and the enhanced catalytic performance of cascaded enzymes, our colorimetric assay system exhibits excellent sensitivity, and the detection limits have been calculated to be 5.2 fM and 0.8 pM for the nucleic acid target (T as a model) and biological substances (ATP as a model), respectively.