Introduction:
Herbicides are at the same time an integral part of modern agricultural technologies and toxic contaminants of consumer products. As well as 2,4-dichlorophenoxyacetic acid (2,4-D) has been on the top list of herbicides in terms of volume of use for decades, methods for its rapid, sensitive and productive control are extremely in demand. Fluorescence polarization immunoassay (FPIA) seems to be a perspective solution for this purpose. The sensitivity of immunoassays depends on the affinity of a specific antibody and from a labeled competitor. In the case of FPIA, this role is played by a conjugate of an antigen and a fluorophore, the so-called tracer. The aim of the presented study is to synthesize new tracers for 2,4-D immunodetection and to develop sensitive FPIA using them.

Methods:
Chlorinated phenoxyacetic acid compounds (2,4-D; 3,4-D; 2,4,5-T) were activated by carbodiimide/succinimide technique and labeled with different amino derivatives of fluorescein (GAF, EDF, BDF, AMF). The structure of the tracers was confirmed by mass-spectrometry. Polyclonal rabbit antiserum against 2,4-D was from XEMA Company, Ltd, Moscow, Russia.

Results:
The (aminoacetamido)fluorescein (GAF) was found to be the most efficient compound for tracer's preparation. The FPIA of 2,4-D using the 2,4-D-GAF tracer was developed by choosing the best values of tracer concentration and antiserum dilution. Time of the assay is 5 min. The assay can be performed outside laboratory using portable polarization fluorimeter Sentry-200 (Ellie, USA). The limit of detection for 2,4-D is 10 ng/mL being lower than common Maximum Residue Limits (100 ng/mL or 100 μg/kg). The developed FPIA is high specific – no cross-reactivities (CR) with other classes of herbicides and only 1% CR with 2,4,5-T.

Conclusions:
New tracer 2,4-D-GAF could be used for sensitive FPIA of herbicide 2,4-D. The FPIA is quick method without separation and wash steps as in ELISA.

Conflicts of interest:
The authors declare that they have no conflict of interest in the publication of this article. The authors have no conflicts of interest to report in this work. Abstract was not submitted elsewhere and was first published here.

Funding:
The study was supported by the Russian Science Foundation; grant number 24-43-00196 (https://rscf.ru/project/24-43-00196/).