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An aptamer biosensor for leukemia marker mRNA detection based on polymerase-assisted signal amplification and aggregation of illuminator

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Abstract

A novel electrochemical luminescence (ECL) aptamer biosensor via polymerase amplification is constructed for label-free detection of leukemia marker mRNA (miR-16). In order to achieve the ultrasensitive detection of the target mRNA, the cyclic target chain displacement polymerization of leukemia marker mRNA assisted with Klenow fragment of DNA polymerase is employed. The determination is carried out by recording the ECL emission of pyridine ruthenium (Ru(bpy)32+) complexes embedded into the assistance DNA (ADNA) loaded on the nanogold surface, after the hybridization reaction between the probe DNA (PDNA) and the remaining sequence of the CP’s stem part, and the formation of a core-shell sun-like structure. The mercapto-modified capture DNA (CP) is immobilized on the surface of a magneto-controlled glassy carbon electrode by Au-S bond. The CP is opened and hybridized with the target mRNA to form double-stranded DNA. In the presence of polymerase, primer DNA, and bases (dNTPs), the primer chain gets access to its complementary sequence of the stem part and then triggers a polymerization of the DNA strand, leading to the release of mRNA and starting the next polymerization cycle. Finally, the composite of PDNA-covered and ADNA-covered (embedded with Ru(bpy)32+) gold nanoparticles (hereafter called AuNPs@(PDNA+ADNA-Ru(bpy)32+) is added, and the ECL intensity is recorded. Because of the polymerization cycle and the aggregation of the illuminator of Ru(bpy)32+, the detected signal is amplified significantly. The results showed that the corresponding ECL signal has a good linear relationship with a logarithm of target mRNA concentration in the range of 1 × 10−16 to 1 × 10−7 mol/L, with a detection limit of 4.3 × 10−17 mol/L. The mRNA spiked in the human serum sample is determined, and the recoveries are from 97.2 to 102.0%. This sensor demonstrates good selectivity, stability, and reproducibility.

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Funding

This research is financially supported by the National Natural Science Foundation of China (No.21765006), the Guangxi Natural Science Foundation, China (No. 2015GXNSFFA139005), and the High-Level Innovation Teams of Guangxi Colleges and Universities and Outstanding Scholars Program (Guijiaoren[2014]49).

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Authors and Affiliations

  1. Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guilin, 541004, China

    Meng Zhang, Fenyue Zhou, Dongli Chen, Hong Hai & Jianping Li

  2. College of Biological Sciences, University of California – Davis, Davis, CA, 95616, USA

    Deqi Zhou

Authors
  1. Meng Zhang
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  2. Fenyue Zhou
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  3. Deqi Zhou
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  4. Dongli Chen
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  5. Hong Hai
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Correspondence to Hong Hai or Jianping Li.

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The authors declare that they have no conflict of interest.

Ethical standards and informed consent

The study and experimental sections were approved by the Hospital of Guilin University of Technology. Human serum samples used in this study do not have any identifying information about all the participants that provided written informed consent.

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Zhang, M., Zhou, F., Zhou, D. et al. An aptamer biosensor for leukemia marker mRNA detection based on polymerase-assisted signal amplification and aggregation of illuminator. Anal Bioanal Chem 411, 139–146 (2019). https://doi.org/10.1007/s00216-018-1424-9

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  • DOI: https://doi.org/10.1007/s00216-018-1424-9

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