Preparation of amyloid N-terminal nonapeptide imprinted monolithic column and evaluation of adsorption properties

Abstract

A novel β-amyloid protein capillary microextraction column was designed and prepared using epitope molecular imprinting technology for specific recognition of trace β-amyloid proteins in complex biological matrices. Using N-terminal nonapeptide of β-amyloid protein as template molecule, choline chloride-MAA and N-hydroxymethyl acrylamide as functional monomers, ethylene glycol dimethacrylate as crosslinker, the imprinted capillary monolithic column was prepared by thermal polymerization in the acetonitrile-water system. The optimal preparation parameters were obtained with the ratio of template: functional monomer: crosslinker at 1:6:16 (mmol/mmol/mmol). The physical property evaluation through Scanning electron microscopy, Fourier transform infrared spectroscopy analysis, zeta potential analysis, particle size analysis, and Brunauer-emmett-teller showed that a porous imprinted monolithic column with a high specific surface area was successfully prepared. The static adsorption experiment showed that the theoretical maximum adsorption capacity was 0.060 μg/column and the optimal imprinting factor was 2.27. Under the optimal extraction conditions, the imprinted column exhibited good template selectivity and excellent robustness. Finally, the extraction efficiency of the capillary imprinted column in actual plasma was evaluated using ELISA method. For Aβ 40 and Aβ 42, the detection limits were 1.00 pg/mL and 0.67 pg/mL, the quantification limits were 3.00 pg/mL and 2.00 pg/mL, the detection ranges were 7.5–120.0 pg/mL and 5.0–80.0 pg/mL, respectively. After extraction of plasma samples, the measured concentrations of Aβ 40 and Aβ 42 by imprinted column were 157.31 pg/mL and 48.22 pg/mL, respectively, which were significantly higher than the measured concentrations by non-imprinted column of 89.60 pg/mL and 41.02 pg/mL. In summary, the epitope imprinted capillary monolithic column prepared in this study can effectively enrich and separate trace amounts of amyloid proteins in complex samples, and is expected to provide a new sample pretreatment method for clinical detection of amyloid proteins.