This study presents a comprehensive computational and experimental investigation into the dual-template molecular imprinting mechanism for catechin (CA) and theophylline (TPH) using peanut twin-like NiFe2O4/CoFe2O4/NCDs nanospheres as a recognition matrix. Density functional theory (DFT) calculations were employed to screen optimal functional monomers and determine the ideal molar ratio between the two template molecules and the monomer. A total of five candidate monomers—L-arginine, o-aminophenol, o-phenylenediamine, salicylic acid, and acrylamide—were evaluated based on their binding energies with CA and TPH. DFT simulations revealed that L-arginine exhibited the lowest binding energy when used in a 1:2:5 molar ratio of TPH:CA:L-arginine, indicating superior complex stability. Natural bond orbital (NBO) analysis confirmed that the oxygen atoms in carbonyl groups and hydroxyl groups, along with nitrogen atoms in amine groups, serve as primary interaction sites due to their negative charge distribution. Molecular electrostatic potential (MEP) maps further visualized these favorable interaction regions.TXNDC5 Antibody Formula Based on these theoretical predictions, an in-situ electrochemical polymerization method was applied to fabricate the dual-template MIP film directly on the NiFe2O4/CoFe2O4/NCDs/GCE surface. The resulting sensor demonstrated highly specific recognition toward both CA and TPH, attributed to the formation of complementary cavities with precise spatial and chemical matching. Electrochemical characterization via cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) confirmed enhanced electron transfer kinetics and increased effective surface area.R-Spondin Antibody web The imprinted sensor showed significantly higher response currents compared to non-imprinted controls, especially after template elution and re-binding.PMID:34751824 Binding constant analysis revealed strong affinity (Kd1 = 1.48 mol L⁻¹ for CA; Kd3 = 2.31 mol L⁻¹ for TPH), while imprinting factor values (IF = 8.45 for CA, IF = 4.37 for TPH) confirmed high selectivity over structural analogs such as quercetin, rutin, morin hydrate, caffeine, xanthine, and theobromine. The sensor also displayed excellent anti-interference performance in the presence of common ions and organic compounds. Practical application in real tea and urine samples yielded recoveries between 98.22% and 104.76%, validating its reliability. This work demonstrates how DFT-guided rational design can effectively optimize dual-template MIP systems, offering a powerful strategy for developing high-performance sensors for simultaneous detection of multiple bioactive compounds.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com