Palindrome design-guided "single-flap-double-duties" and "dual-enzymes-quadruple-cycles" enabled nano-signal...

Flap endonuclease 1 (FEN1), a structure-specific nuclease, is usually overexpressed in various types of cancer cells and has been recognized as a promising biomarker for molecular diagnostics. In this work, we developed a one-tube and ultrasensitive method for FEN1 sensing based on customized target recognition, exponential signal amplification and nano-signal transduction. Due to rational palindrome design, only two DNA substrates and a pair of enzymes were needed in the sensing. Within the dumbbell DNA probe, the palindromic sequence was divided into two parts. One was designed as 5'-end overhanging flap that can trigger signal amplification in presence of FEN1 and prevent signal output in absence of FEN1. And the other one was complementary sequence of 5' flap which is available for initiating quadruple isothermal-amplification cycles assisted with a palindromic hairpin and driven by polymerase and endonuclease. The seamless integration of "single-flap-double-duties" with "dual-enzymes-quadruple-cycles" thus enabled exponential signal amplification of fluorescent copper nanoparticles and contributed to ultrasensitive FEN1 sensing with a detection limit of 3.5 × 10^-6 U/mL. Thanks to its high selectivity and anti-interference ability, this method was able to unambiguously distinguish cancer cells from normal cell based on the test results of cellular FEN1. This work is expected to provide a promising strategy of cancer biomarker detection for advanced molecular diagnostics.