Development of an Immunofluorescent Capillary Sensor for the Detection of Zearalenone Mycotoxin.

Abstract

A capillary-based immunofluorescence sensor was developed and incorporated in a flow injection analysis system. The light-guiding capillary was illuminated axially by a 473 nm/5 mW solid state laser through a tailored optofluidic connector. High sensitivity of the system was achieved by efficiently collecting and detecting the non-guided fluorescence signal scattered out along the wall of the capillary. The excitation was highly suppressed with bandpass and dichroic filters by simultaneously exploiting the guiding effect inside the capillary. The glass capillary used as a measuring cell was silanized in liquid phase by 3-aminopropyltriethoxysilane (APTS), and the biomolecules were immobilized using glutaraldehyde inside the capillary. The applicability of the developed system was tested with a bovine serum albumin (BSA)-anti-BSA-IgG model-molecule pair, using a fluorescently labeled secondary antibody. Based on the results of the BSA-anti-BSA experiments, a similar setup using a primary antibody specific for zearalenone (ZON) was established, and a competitive fluorescence measurement system was developed for quantitative determination of ZON. For the measurements, 20 µg/mL ZON-BSA conjugate was immobilized in the capillary, and a 1:2500 dilution of the primary antibody stock solution and a 2 µg/mL secondary antibody solution were set. The developed capillary-based immunosensor allowed a limit of detection (LOD) of 0.003 ng/mL and a limit of quantification (LOQ) of 0.007 ng/mL for ZON in the competitive immunosensor setup, with a dynamic detection range of 0.01-10 ng/mL ZON concentrations.