On-line monitoring of the dopamine-based molecular imprinting processes for protein templates with the assistance of a fluorescent indicator

Abstract

On-line monitoring of the dopamine (DA)-based molecular imprinting processes over Fe₃O₄@SiO₂-NH₂ nanoparticles (SiMNPs) is reported by using a real-time quantitative PCR machine. Taking advantages of the efficient fluorescence quenching capability of polydopamine (PDA) and its high binding affinity to rhodamine B (RhB), we performed molecular imprinting against different proteins with free dopamine as the functional monomer and RhB as a fluorescent indicator. Along with the template molecules, the fluorescent indicators were continuously encapsulated into the PDA layer formed on the surface of the SiMNPs, resulting in immediate quenching of the fluorescence, which can be conveniently monitored in real time. As proteins showed sequence-dependent influences on the oxidation of dopamine and subsequent self-assembly on the surface of the SiMNPs, the observed fluorescence signals clearly indicated the polymerization progress in the presence of the template proteins, allowing precise control of the reaction time for different templates at a given initial concentration. The optimum end point of the reaction was found to be when 90 ± 3% of the templates had been encapsulated into the polymer, which offered the highest imprinting factor and selectivity. We applied the approach to prepare a primary PDA-based surface imprinted polymer for a multifunctional protein apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1). After further introduction of 3-hydroxyphenylboronic acid to the interfaces between APE1 and PDA, the resultant molecularly imprinted polymers (MIP-II) enabled quantitative isolation APE1 from cell lysate samples. The developed approach will be useful for the quantitative preparation of PDA-based MIPs for precious template proteins with limited input quantity. It is also applicable for further study on the effects of different proteins or peptides on the PDA formation reactions.

Keywords: Dopamine polymerization; Fluorescence; Molecular imprinting; On-line monitoring; Rhodamine B; Template protein.