Microchimica Acta

, 186:840 | Cite as

Multiplexed aptasensing of food contaminants by using terminal deoxynucleotidyl transferase-produced primer-triggered rolling circle amplification: application to the colorimetric determination of enrofloxacin, lead (II), Escherichia coli O157:H7 and tropomyosin

  • Yumei Du
  • Yangyang Zhou
  • Yanli Wen
  • Xiaojun Bian
  • Yuanyuan Xie
  • Weijia Zhang
  • Gang LiuEmail author
  • Juan YanEmail author
Original Paper


A colorimetric assay is described for simultaneous detection of multiple analytes related to food safety. It is based on the use of a sandwich aptasensor and terminal deoxynucleotidyl transferase (TdT) which produces a primer for subsequent rolling circle amplification (RCA). Two split aptamer fragments (Apt1 and Apt2) are firstly immobilized, Apt1 on gold nanoparticles (AuNPs), and Apt2 on magnetic beads (MBs). They are then used in a sandwich aptasensor. In the presence of analyte, two probes could specifically recognize target and form a ternary assembly, and the magnetic beads also act to separate rapidly and enrich the target. Then, the extension of template-free DNA is triggered by TdT at the exposed 3′-hydroxy terminals of Apt1. This produces polyA sequences that serve as primers for subsequent RCA. The product of RCA is hybridized with a complementary horse radish peroxidase (HRP) DNA probe. HRP catalyzes the H2O2-mediated oxidation of tetramethylbenzidine (TMB) and forms a blue chromogenic product. After magnetic separation, the absorption values of the blue product in the supernatant are measured at a wavelength of 600 nm. Based on this dual amplification mechanism, the assay was applied to multiplexed determination of enrofloxacin (ENR), lead(II), Escherichia coli O157:H7 and tropomyosin. Exemplarily, ENR is detectable at concentrations down to 2.5 pg mL−1 with a linear range that extends from 1 pg mL−1 to 1 μg·mL−1. The assay was validated by analysis of spiked fish samples. Recoveries range between 87.5 and 92.1%.

Graphical abstract

Schematic representation of a TdT-RCA based aptasensor for multiple analytes related to food safety. It makes use of sandwich aptasensors and TdT-produced universal primer-triggered RCA reaction. dATP: deoxyadenosine triphosphate, TdT: Terminal Deoxynucleotidyl Transferase, RCA: rolling circle amplification, TMB: 3,3′,5,5’-Tetramethylbenzidine.


Signal amplification method Isothermal nucleic acid amplification Food safety detection Biodetection Gold nanoparticles Magnetic beads Aptasensor Antibiotics 



This work was financially supported by the National Natural Science Foundation of China (21775102, 21775104), National Key R&D Program of China (2017YFF0204603, 2018YFF0212803) and Shanghai Rising-Star Program (16QB1403100).

Compliance with ethical standards

Conflict of interest

This article does not contain any studies involving human participants performed by any of the authors.

Supplementary material

604_2019_3935_MOESM1_ESM.docx (1.1 mb)
ESM 1 (DOCX 1174 kb)


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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture; Shanghai Engineering Research Center of Aquatic-Product Process & Preservation; College of Food Science and TechnologyShanghai Ocean UniversityShanghaiChina
  2. 2.Laboratory of Biometrology, Division of Chemistry and Ionizing Radiation Measurement TechnologyShanghai Institute of Measurement and Testing TechnologyShanghaiChina
  3. 3.Fifth People’s Hospital of Shanghai and Institutes of Biomedical Sciences, and State Key Laboratory of Molecular EngineeringFudan UniversityShanghaiChina

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