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The DNA microarray has distinctive advantages of high-throughput and less complicated operations, but tends to have a relatively low sensitivity. Catalytic hairpin assembly (CHA) is one of the most promising enzyme-free, isothermal DNA circuit for high efficient signal amplification. Here, a microarray-based catalytic hairpin assembly (mi-CHA) biosensing method has been developed to detect various miRNAs in a single test simultaneously. The target miRNA can trigger conformational transformations of hairpin-structured DNA probes on the chip surface and lead to the specific signal amplification. A significant advantage of this approach is that each duplex produced by the solid-phase CHA will be immobilized on the certain location of the chip and release fluorescent signal via the universal domain, eliminating the requirement of different fluorophores. This method has manifested a high detection sensitivity of human cancer-associated miRNAs (miR-21 and miR-155) down to 1.33 fM and promised a high specificity to distinguish single-base mismatches. Furthermore, the practicability of this method was demonstrated by analyzing target miRNAs in human serum and cancer cells. The experimental results suggest that the proposed method has high-throughput analytical potential and could be applied to many other clinical diagnosis.
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•A novel enzyme-free isothermal amplification method has been developed for simultaneous detection of various miRNAs in a single test, and achieved the LOD of 1.33 fM.•The practicability of this method was supported by analyzing target miRNAs in human serum and cancer cells.•The modular design of CHA components allows using the same fluorescent probe for different miRNA targets.