ABSTRACT
Treatment of Alzheimer's disease (AD) is plagued by a lack of practical and reliable methods allowing early diagnosis of the disease. We here demonstrate that robust receptors prepared by molecular imprinting successfully address current limitations of biologically derived receptors in displaying affinity for hydrophobic peptide biomarkers for AD under denaturing conditions. C-terminal epitope-imprinted polymers showing enhanced binding affinity for Aß1-42 were first identified from a 96-polymer combinatorial library. This information was then used to synthesize molecularly imprinted polymers for both of the ß-amyloid (Aß) isoforms and a corresponding nonimprinted polymer. A solid-phase extraction method was developed to be compatible with sample loading under conditions of complete protein denaturation. This resulted in a method capable of quantitatively and selectively enriching a shorter C-terminal peptide corresponding to the sequences Aß33-40 and Aß33-42 as well as the full-length sequence Aß1-40 and Aß1-42 from a 4 M guanidinum chloride solution. Application of the method to serum allowed selective, high-recovery extraction of both biomarkers at spiking levels marginally higher than clinically relevant concentrations found in cerebrospinal fluid.
Subject(s)
Alzheimer Disease , Amyloid beta-Peptides/blood , Amyloid beta-Peptides/chemistry , Blood Chemical Analysis/methods , Molecular Imprinting , Polymers/chemical synthesis , Protein Denaturation , Amino Acid Sequence , Biomarkers/blood , Humans , Molecular Sequence Data , Peptide Fragments/blood , Peptide Fragments/chemistryABSTRACT
A comprehensive comparison of five chromatographic stationary phases based on molecularly imprinted polymers is presented. Efficiency, imprinting factors, water compatibility and batch-to-batch reproducibility are discussed for crushed monolith, microspheres, two silica-based composites and capillary monoliths, all imprinted with the local anaesthetic bupivacaine. Synthesis protocol and chromatographic test conditions have been kept fixed within certain limits, in order to provide further insight into the strengths and weaknesses of the different formats. Excluding microparticles, all formats give satisfactory performance, especially in aqueous mobile phases. An assessment of batch-to-batch reproducibility in different mobile phases adds further value to this comparison study.
