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Anal Chim Acta. 2015 Jun 16;879:91-6. doi: 10.1016/j.aca.2015.03.030. Epub 2015 Mar 24.

Enzymatic cleavage and mass amplification strategy for small molecule detection using aptamer-based fluorescence polarization biosensor.

Author information

1
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.
2
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.
3
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.
4
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.

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.

KEYWORDS:

Aptamer; Enzymatic cleavage protection; Fluorescence polarization; Protein amplifier; Small-molecule detection

PMID:
26002482
DOI:
10.1016/j.aca.2015.03.030
[Indexed for MEDLINE]

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