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

Content Provider	Springer Nature Link
Author	Guo, Limin Hao, Lihua Zhao, Qiang
Copyright Year	2016
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.
Starting Page	4715
Ending Page	4722
Page Count	8
File Format	PDF
ISSN	16182642
Journal	Analytical and Bioanalytical Chemistry
Volume Number	408
Issue Number	17
e-ISSN	16182650
Language	English
Publisher	Springer Berlin Heidelberg
Publisher Date	2016-04-23
Publisher Place	Berlin, Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Aptamer Thrombin Rolling circle amplification Multiple enzyme labeling Signal amplification Protein detection Analytical Chemistry Biochemistry Laboratory Medicine Characterization and Evaluation of Materials Food Science Environmental Monitoring/Analysis
Content Type	Text
Resource Type	Article
Subject	Analytical Chemistry Biochemistry

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