title: Fluorescent assay for alkaline phosphatase activity based on graphene oxide integrating with λ exonuclease.
abstract: A novel fluorescence turn-on strategy for the alkaline phosphatase (ALP) assay is developed based on the preferential binding of graphene oxide (GO) to single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) coupled with λ exonuclease (λ exo) cleavage. Specifically, in the absence of ALP, the substrate-dsDNA constructed by one oligonucleotide with a fluorophore at the 3'-end (F-DNA) and its complementary sequence modified with a 5'-phosphoryl termini (p-DNA), is promptly cleaved by λ exo, and the resulting F-DNA is adsorbed on GO surface, allowing fluorescence quenching. Whereas the introduction of ALP leads to the hydrolysis of the P-DNA, and the yielding 5'-hydroxyl end product hampers the λ exo cleavage, inducing significant fluorescence enhancement due to the weak binding of dsDNA with GO. Under the optimized conditions, the approach exhibits high sensitivity and specificity to ALP with a detection limit of 0.19 U/L, and the determination of ALP in spiked human serum samples has also been realized. Notably, this new approach not only provides a novel and sensitive platform for the ALP activity detection but also promotes the exploitation of the GO-based biosensing for the detection of the protein with no specific binding element, and thus extending the GO-based sensing applications into a new field.
authors.affiliation: Department of Biology and Chemical Engineering, Nanyang Institute of Technology, Nanyang 473004, China.
authors.forename: Xue-Guo
authors.initials: XG
authors.lastname: Liu
authors.affiliation: College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China. Electronic address: xingshujing2005@163.com.
authors.forename: Xiao-Jing
authors.initials: XJ
authors.lastname: Xing
authors.affiliation: College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Li
authors.affiliation: College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China.
authors.forename: Yong-Ming
authors.initials: YM
authors.lastname: Guo
authors.affiliation: College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China.
authors.forename: Ye-Zhen
authors.initials: YZ
authors.lastname: Zhang
authors.affiliation: College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.affiliation: College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang 473061, China. Electronic address: lfzhang66@126.com.
authors.forename: Lian-Feng
authors.initials: LF
authors.lastname: Zhang
mesh_terms: D000469:Alkaline Phosphatase / Q000097:blood* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D005092:Exonucleases / Q000378:metabolism; D006108:Graphite / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D010087:Oxides / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
chemical_list: D004277:DNA, Single-Stranded; D010087:Oxides; D006108:Graphite; D004247:DNA; D005092:Exonucleases; D000469:Alkaline Phosphatase
keywords: Alkaline phosphatase; Fluorescence; Graphene oxide; λ Exonuclease