Affordable Access

Access to the full text

Recombinase polymerase amplification combined with a magnetic nanoparticle-based immunoassay for fluorometric determination of troponin T

  • Ivanov, Alexandr V.1
  • Safenkova, Irina V.1
  • Zherdev, Anatoly V.1
  • Dzantiev, Boris B.1
  • 1 Research Centre of Biotechnology of the Russian Academy of Sciences, A.N. Bach Institute of Biochemistry, Leninsky Prospect 33, Moscow, 119071, Russia , Moscow (Russia)
Published Article
Microchimica Acta
Publication Date
Jul 18, 2019
DOI: 10.1007/s00604-019-3686-0
Springer Nature


The authors describe a method for the improvement of the sensitivity of immunoassays. This was achieved by a combination of immunoassay and an amplification based on the use of a multiplied DNA probe. The immunoassay was enhanced via recombinant polymerase amplification (RPA). It enables fast (35 min) isothermal multiplication of DNA at 37 °C. This concept was demonstrated for a sandwich immunoassay that is making use of magnetic nanoparticles conjugated to first specific antibodies and then to second specific antibodies linked to reporter DNA via biotin-streptavidin binding. Reporter DNA multiplied by RPA was quantified fluorometrically via the carboxyfluorescein label. Human cardiac troponin T (cTnT), a biomarker for acute myocardial infarction, was used as the target of the immunoRPA (iRPA). The assay can detect cTnT in serum and in plasma within 2 h, with detection limits as low as 12.5 ± 1.1 pg•mL−1 and 9.4 ± 2.1 pg•mL−1, respectively. This represents increased sensitivity and decreased assay time compared to classical ELISAs (3.5 ± 0.3 ng•mL−1) or immuno-PCR (4.3 ± 0.8 ng•mL−1) that were carried out using the same immunoreagents and reporter DNA. The good performance of this iRPA was underlined by its successful application to the analysis of plasma samples. The iRPA has significant advantages relative to other DNA amplification-based immunoassays due to isothermal conditions and analysis at 37 °C. Graphical abstractSchematic representation of a new kind of immunoassay that is enhanced by making use of recombinase polymerase amplification (immunoRPA). Reporter DNA was conjugated with specific antibodies, then amplified and finally quantified fluorometrically. Limit of detection of troponin T in plasma within 2 h was 9.4 ± 2.1 pg•mL−1.

Report this publication


Seen <100 times