Organic polymer dot-based fluorometric determination of the activity of horseradish peroxidase and of the concentrations of glucose and the insecticidal protein toxin Cry1Ab/Ac

Abstract

Fluorescent polymer dots (PDs) with maximum excitation/emission wavelengths of 410/515 nm were prepared in water solution from 1,4-benzoquinone and ethylenediamine. The green fluorescence of these PDs is screened off by the red-colored oxidation product (PPDox, maximum absorption at 510 nm) formed by horseradish peroxidase (HRP)-catalyzed oxidation of p-phenylenediamine (PPD). It causes the reduction of the fluorescence intensity of the PDs due to spectral overlap and an inner filter effect (IFE). If glucose is enzymatically oxidized under the formation of H2O2, the formed H2O2 can be quantified by the above IFE. The assay for HRP activity and glucose have detection limits of 0.2 U·L−1 and 0.1 μM, respectively. The nanoprobe was further extended to an immunosorbent assay (ELISA) for the determination of insecticidal Cry1Ab/Ac protein with a detection limit of 0.25 ng·mL−1. The ELISA was applied to rice leaf analysis.

Schematic representation of fluorometrict enzyme-linked immunosorbent assay for Cry1Ab/Ac protein detection based on horseradish peroxidase (HRP)-triggered fluorescence quenching of polymer dots (PDs). Quenching is caused by an inner filter effect (IFE) caused by PPDox, the oxidation product of p-phenylenediamine (PPD).

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Acknowledgments

This study was funded by the Fundamental Research Funds for the Central Universities, China (2662018JC011 and 2662019PY023).

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Correspondence to Hong Jiang or Jimei Ma.

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Cheng, X., Sun, L., Li, R. et al. Organic polymer dot-based fluorometric determination of the activity of horseradish peroxidase and of the concentrations of glucose and the insecticidal protein toxin Cry1Ab/Ac. Microchim Acta 186, 731 (2019). https://doi.org/10.1007/s00604-019-3831-9

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Keywords

  • Nanoprobe
  • p-Phenylenediamine
  • Glucose oxidase
  • Inner filter effect
  • Fluorescent enzyme-linked immunosorbent assay
  • Rice leaves