Ultrasensitive fluorometric oligonucleotide immunoassay for the simultaneous and efficient detection of two mycotoxins in agricultural products

Abstract

An ultrasensitive fluorometric oligonucleotide immunoassay (UFOIA) based on a fluorometric oligonucleotide and magnetic separation was proposed for the simultaneous detection of two mycotoxins. Two kinds of magnetic nanoparticle (MNP) probes and their corresponding fluorometric oligonucleotide probes were prepared. After the immune reaction, Cy5-linked and 6-FAM-linked oligonucleotides were dissociated and applied to detect fluorescence signals simultaneously. Under optimal conditions, the detection ranges of the UFOIA were in the range of 0.654–1438.8 pg mL⁻¹ for zearalenone (ZEN) and 0.215–3190.1 pg mL⁻¹ for aflatoxin B₁ (AFB₁). The limits of detection (LODs) were 0.378 pg mL⁻¹ for ZEN and 0.043 pg mL⁻¹ for AFB₁, which showed improved sensitivities of 529-fold and 112-fold compared to those from the ELISA. The positive results of the UFOIA for authentic agricultural products were highly correlated with those from LC-MS/MS. The specificity, accuracy, precision and reliability of the UFOIA are well demonstrated. The proposed UFOIA method achieved the simultaneous and ultrasensitive detection of mycotoxins at the pg mL⁻¹ level, which was a considerable improvement. This study might provide an alternative approach for detecting multi-component contamination equipped with the notable highlights of ultrasensitivity, simultaneity, simplicity, high efficiency and a low background signal.