Molecular Simulation-Guided Development of a Ractopamine Aptasensor Based on Magnetic Microspheres.

β-Adrenergic agonists are employed in the livestock industry to promote the growth of poultry and livestock. However, due to the frequent lack of scientific guidance in medication administration among farmers, issues such as indiscriminate use, misuse, and noncompliance with withdrawal periods have arisen. These practices result in drug residues, triggering food safety concerns and posing a threat to consumer health. Consequently, there is an urgent need to develop methods for detecting the illicit use of drugs in the livestock industry. In this study, a colloidal gold test strip sensor for the detection of ractopamine was developed based on nucleic acid aptamers, magnetic microspheres, and isothermal amplification techniques. Using isophorone diisocyanate as the monomer, polyurea microspheres with uniform and adjustable particle sizes were successfully prepared via precipitation polymerization. Subsequently, carboxyl-functionalized magnetic microspheres with a layer-by-layer assembly structure were obtained. The carboxyl-functionalized magnetic microspheres exhibited excellent dispersibility and superparamagnetism, with a particle size of approximately 2 µm. They demonstrated a favorable adsorption effect on bovine serum albumin, with an adsorption capacity of 10-14 mg g. Through computer simulations, molecular docking, and structural optimization, the sequence of a published ractopamine aptamer was refined, and an aptamer with superior binding ability and specificity compared to the original one was identified. The isothermal amplification system was also improved. An innovative isothermal amplification method was proposed, which involved fixing the template amount in isothermal amplification and establishing a linear relationship between primer and amplification strand concentrations. The template and primer sequences for isothermal amplification were designed, and factors such as amplification temperature, time, and template addition amount were optimized to determine the optimal conditions. When the designed aptamer sensor was used to detect ractopamine, it showed a detection limit of 62.5 µM, indicating favorable detection performance. Currently, there are no relevant studies reported domestically or internationally on the preparation and application of layer-by-layer assembled carboxyl microspheres with polyurea as the core, combined with aptamer sensors for detection purposes. Therefore, the successful development of this sensor detection method provides a valuable reference for research on biosensors combining magnetic microspheres and aptamers.