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An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection

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Abstract

We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1 pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications.

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Acknowledgments

We thank the support from National Natural Science Foundation of China (Grant No. 21222503) and Outstanding Youth Talents Program of Shanxi Province.

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Authors and Affiliations

  1. Institute of Environmental Science, College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan, Shanxi, 030006, China

    Limin Guo, Lihua Hao & Qiang Zhao

  2. School of Chinese Materia Medica, Shanxi University of Traditional Chinese Medicine, Taiyuan, Shanxi, 030024, China

    Limin Guo

  3. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China

    Lihua Hao & Qiang Zhao

Authors
  1. Limin Guo
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  2. Lihua Hao
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  3. Qiang Zhao
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Correspondence to Qiang Zhao.

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Guo, L., Hao, L. & Zhao, Q. An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection. Anal Bioanal Chem 408, 4715–4722 (2016). https://doi.org/10.1007/s00216-016-9558-0

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  • DOI: https://doi.org/10.1007/s00216-016-9558-0

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