title: High sensitive and label-free colorimetric DNA detection based on nicking endonuclease-assisted activation of DNAzymes.
abstract: Horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) attracts growing interest as an amplifying label for biorecognition and biosensing events, especially for DNA detection. However, in the traditional designs, one target molecule can only generate one HRP-DNAzyme, which limits the signal enhancement and thus its sensitivity. In this article, we propose an amplified and label-free colorimetric DNA detection strategy based on nicking endonuclease (NEase)-assisted activation of HRP-DNAzymes (NEAA-DNAzymes). This new strategy relies on the hairpin-DNAzyme probe and NEase-assisted target recycling. In the hairpin-DNAzyme probe, the HRP-DNAzyme sequence is protected in a "caged" inactive structure, whereas the loop region includes the target complementary sequence. Upon hybridization with target, the beacon is opened, resulting in the activation of the HRP-DNAzyme. Meanwhile, upon formation of the duplex, the NEase recognizes a specific nucleotide sequence and cleaves the hairpin-DNAzyme probe into two fragments. After nicking, the fragments of the hairpin-DNAzyme probe spontaneously dissociate from the target DNA. Amplification is accomplished by another hairpin-DNAzyme probe hybridizing to the released intact target to continue the strand-scission cycle, which results in activation of numerous DNAzymes. The activated HRP-DNAzymes generate colorimetric or chemiluminescence readout signals, thus providing the amplified detection of DNA. The detection limit of the colorimetric method is 10 pmol/L, which are three orders of magnitude lower than that without NEase. In addition, the detection limit of the chemiluminescence method is 0.2 pmol/L. Meanwhile, this strategy also exhibits high discrimination ability even against single-base mismatch.
authors.affiliation: The Key Lab of Analysis and Detection Technology for Food Safety of the MOE, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou 350002, PR China.
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Qiu-Hong
authors.initials: QH
authors.lastname: Yao
authors.forename: Hua-E
authors.initials: HE
authors.lastname: Fu
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Zhang
authors.forename: Huang-Hao
authors.initials: HH
authors.lastname: Yang
mesh_terms: D003124:Colorimetry / Q000379:methods*; D004247:DNA / Q000032:analysis*; D021881:DNA, Catalytic / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004789:Enzyme Activation; D006735:Horseradish Peroxidase; D057230:Limit of Detection; D008163:Luminescent Measurements; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization
chemical_list: D021881:DNA, Catalytic; D004247:DNA; D006735:Horseradish Peroxidase; D003850:Deoxyribonuclease I