Neonicotinoid Microsphere Immunosensing for Profiling Applications in Honeybees and Bee-Related Matrices

Neonicotinoids are the most commonly used insecticides due to their effectiveness. However, non-targeted insects, especially bees, are also affected by neonicotinoids. Therefore, neonicotinoid application can contribute to the declining bee populations worldwide. The presented study describes the development of novel competitive, fluorescent microsphere-based suspension immunoassays for neonicotinoid profiling and their application to bees and essential bee-related matrices, using the Multi-Analyte Profiling (xMAP) technology. For the construction of these neonicotinoid microsphere immunoassays (nMIAs), neonicotinoid-ovalbumin conjugates were coupled to unique fluorescent, paramagnetic microspheres, which competed with the free neonicotinoids that were present in test samples for interacting with the corresponding, specific antibodies. In total, five independent nMIA's were developed for the detection of imidacloprid, acetamiprid, clothianidin, thiacloprid, thiamethoxam, dinotefuran, nitenpyram and imidaclothiz with the limits of detection being for 0.01 ng/mL, 0.01 ng/mL, 0.02 ng/mL, 0.02 ng/mL, 0.003 ng/mL, 2.95 ng/mL, 0.09 ng/mL and 0.04 ng/mL, respectively. The developed nMIAs were applied to fortified matrices including surface water, pollen, honey and honeybees. All of the neonicotinoids, except dinotefuran, could be sensitively detected in all of the tested environmental matrices and bees, with there being sensitivities of 1 ng/mL in water and 10 ng/g in solid materials. These nMIAs provide a rapid profiling method for all of the common neonicotinoids, including those that are banned by the European Union for outdoor use. The developed method can contribute to healthy and sustainable beekeeping, globally, via its application in the apiary environment.