Nanomagnetic biomimetic polymers with restricted access (magnetic RAMIP) obtained by semicovalente and non-covalent synthesis aiming application in immunoassays and immunosensors

Author(s)

Rafael Rovatti Pupin

• Document type: Master's Dissertation
• Press: Araraquara. 2017-08-29.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Química. Araraquara
• Defense date: 2017-08-04
• Advisor: Maria del Pilar Taboada Sotomayor; María Isabel Pividori

Abstract

Biotin (vitamin B7) can be attached with different types of molecules and still be selectively recognized by avidin/streptavidin proteins due to the high affinity constant between them (Ka = 1,0×1015 L mol-1). Thus, this interaction provides an excellent increase in the sensitivity of analyzes; which leads to the development of analytical methods for the determination of different biotinylated compounds. The use of biological materials, such as antibodies or proteins, in analyzes is often expensive and has low stability of biomolecules under adverse conditions, which has led to the synthesis of materials that can mimic the biological interactions of recognition; which has been successfully made by molecularly printed polymers (MIP). These polymers have advantages over biomolecules because they have low cost, easy preparation and high mechanical and thermal resistance. Thus, this dissertation explores the synthesis, characterization, optimization and application of different MIP that has biotin as the target molecule. Initial studies focused on the synthesis of magnetic MIP (MMIP) with restricted-access (RAMIP) to evaluate protein exclusion by modifying the polymer with bovine serum albumin (BSA) and polyethylene glycol (PEG). While RAMIP coated with BSA didn’t present good results in the experiments, the RAMIP coated with PEG, after optimization, presented highly satisfactory results in protein exclusion (with values ranging from 98 to 99.4% of protein exclusion) and good results in the rebiding of biotin (2.0 mg of biotin per gram of polymer). The second part of the work was focused on the study of influence of molecular imprinting (non-covalent and semi-covalent) on the capacity of the selective recognition of MMIP. For this, the analyte-monomer complex was previously synthesized (by a covalent bond between biotin and allyl alcohol as functional monomer). This MMIP was submitted to the biotin recognition assays using non-covalent interactions, being nominated as semi-covalent MMIP; whose results were compared with non-covalent biotin MMIP previously optimized. The semi-covalent MMIP had an affinity constant for biotin of 5,9×104 L mol-1, while for non-covalent MMIP it was 2,4×103 L mol-1. Finally, MIP and MMIP for biotin (synthesized via non-covalent) were applied in biomimetic assays using ELISA and Lateral Flow techniques. In the ELISA assays, it was evaluated MMIP recognition capacity by biotin-HRP and biotinylated DNA (of E. coli and Salmonella bacteria). For biotin-HRP, MMIP showed good retention results (in the range of 5.0 to 2500 ng mL-1), and good signal intensity. For biotinylated DNA of both bacteria, MMIP showed higher retention of the amplicon when compared to MNIP. The application of MIP in Lateral Flow devices it wasn’t reported before. Thus, after optimizing all the parameters to design the test line containing MIP (as solvent, mass, solutions, etc.), the system's response to different biotinylated molecules was evaluated: for BSA-biotin and Atto 655-biotin dye the results were not satisfactory; for biotin-HRP, biotin 4-fluorescein and the amplicons MIP showed good selective recognition response. Thus, the imprinted polymers are promising materials in the development of biomimetic assays. (AU)

• FAPESP's process: 15/04367-1 - Nanomagnetic biomimetic polymers with restricted access (magnetic RAMIP) obtained by semicovalente and non-covalent synthesis aiming application in immunoassays and immunosensors
• Grantee: Rafael Rovatti Pupin
• Support Opportunities: Scholarships in Brazil - Master