Strand Displacement-Induced Enzyme-Free Amplification for Label-Free and Separation-Free Ultrasensitive Atomic Fluorescence Spectrometric Detection of Nucleic Acids and Proteins

In previous work, we have developed a simple strategy for a label-free and separation-free bioassay for target DNA and protein, with the limit of detection at the nM level only. Herein, taking advantage of atomic fluorescence spectrometric detection of metal ions and amplification of DNA, a label-free and separation-free ultrasensitive homogeneous DNA analytical platform for target DNA and protein detection was developed on the basis of an enzyme-free strand displacement signal amplification strategy for dramatically improved detectability. Using the T-Hg2+-T hairpin structure as the probe, the target DNA binds with HP (T-Hg2+-T hairpin structure) and released the Hg2+ first; then, the P4 (help DNA) hybridizes with target-P3 complex and free the target DNA, which is used to trigger another reaction cycle. The cycling use of the target amplifies the mercury atomic fluorescence intensity for ultrasensitive DNA detection. Moreover, the enzyme-free strand displacement signal amplification analytical system was further extended for protein detection by introducing an aptamer-P2 arched structure with thrombin as a model analyte. The current homogeneous strategy provides an ultrasensitive AFS detection of DNA and thrombin down to the 0.3 aM and 0.1 aM level, respectively, with a high selectivity. This strategy could be a promising unique alternative for nucleic acid and protein assay.