title: Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence polarization biosensor.
abstract: Fluorescence polarization (FP) assays incorporated with fluorophore-labeled aptamers have attracted great interest in recent years. However, detecting small molecules through the use of FP assays still remains a challenge because small-molecule binding only results in negligible changes in the molecular weight of the fluorophore-labeled aptamer. To address this issue, we herein report a fluorescence polarization (FP) aptamer assay that incorporates a novel signal amplification strategy for highly sensitive detection of small molecules. In the absence of adenosine, our model target, free FAM-labeled aptamer can be digested by nuclease, resulting in the release of FAM-labeled nucleotide segments from the dT-biotin/streptavidin complex with weak background signal. However, in the presence of target, the FAM-labeled aptamer-target complex protects the FAM-labeled aptamer from nuclease cleavage, allowing streptavidin to act as a molar mass amplifier. The resulting increase in molecular mass and FP intensity of the aptamer-target complex provides improved sensitivity for concentration measurement. The probe could detect adenosine from 0.5 μM to 1000 μM, with a detection limit of 500 nM, showing that the sensitivity of the probe is superior to aptamer-based FP approaches previously reported for adenosine. Importantly, FP could resist environmental interferences, making it useful for complex biological samples without any tedious sample pretreatments. Our results demonstrate that this dual-amplified, aptamer-based strategy can be used to design fluorescence polarization probes for rapid, sensitive, and selective measurement of small molecules in complicated biological environment.
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Kang
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China. Electronic address: xbzhang@hnu.edu.cn.
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Cui
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China.
authors.forename: Hongmin
authors.initials: H
authors.lastname: Meng
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Mei
authors.affiliation: Department of Chemistry and Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Shands Cancer Center, University of Florida, Gainesville, FL 32611-7200, USA.
authors.forename: Cuichen
authors.initials: C
authors.lastname: Wu
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China.
authors.forename: Songlei
authors.initials: S
authors.lastname: Ren
authors.affiliation: Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha, Hunan 410082, China; Department of Chemistry and Physiology and Functional Genomics, Center for Research at the Bio/Nano Interface, Shands Cancer Center, University of Florida, Gainesville, FL 32611-7200, USA.
authors.forename: Weihong
authors.initials: W
authors.lastname: Tan
mesh_terms: D000241:Adenosine / Q000032:analysis*; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D017839:Crotalus; D005454:Fluorescence Polarization / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D057230:Limit of Detection; D043264:Phosphodiesterase I / Q000378:metabolism; D030162:Reptilian Proteins / Q000378:metabolism
chemical_list: D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D030162:Reptilian Proteins; D043264:Phosphodiesterase I; D000241:Adenosine
keywords: Aptamer; Enzymatic cleavage protection; Fluorescence polarization; Protein amplifier; Small-molecule detection