Study On A Biomimetic Enzyme-tinked Immunosorbent Assay (BELISA) Method With A Hydrophilic Imprinted Membrane For Fast Determination Of Trace Acrylamide

Acrylamide （AA） is an organic monomer monocase to synthesize polyacrylamide andother polymers. Generally speaking, it is produced in starch-based foods during thedeep-heating process. Toxicological experiments show that acrylamide is neurotoxicity,carcinogenicity and reproductive-development toxicity. In2002, researchers announced thathigh levels of acrylamide had been found in food products. So it is a hotspot on food safety tostrengthen the detection and controlment approaches of acrylamide and its formationmechanism, toxicological characteristics, detection method and protective measure haveattracted worldwide concern.Biomimetic enzyme-linked immunosorbent assay （BELISA） method with a hydrophilicimprinted membrane for the rapid determination of acrylamide in food was founded in thisresearch. The main results are as follows:（1） Preparation of the acrylamide haptens and biomimetic antibody. Four kinds ofacrylamide haptens were designed respectively with double bond and amido link as reactivegrounds and0C、3C、8C as the optimal linking group. The haptens were synthesized byreaction of alkyl chain with different atoms and acryloyl chloride. Using HorseradishPeroxidase （HRP）, enzyme conjugates were prepared by active ester method andglutaraldehyde method. The novel imprinted membrane was directly synthesized on the wellsurface of MaxiSorp polystyrene96-well plate in aqueous environment using water/acetonitrile （v/v,2:3） as solution, acrylamide as the template, methacrylic acid （MAA） as thefunctional monomer, ethyleneglycol dimethacrylate （EGDMA） as the cross-linker,2，2-Azobisisobutyronitrile （AIBN） as evocating agent. The scanning electron microscope （SEM）images showed that the membrane possessed uniform and smooth surface.（2） BELISA conditions optimization. Results showed that the enzyme conjugate whichchose amido link as reactive grounds and8C as the optimal linking group had a broaderinhibition to acrylamide than the other enzyme conjugates. Parameters including the enzymeconjugate concentration, preparing solvent composition and pH were also optimized in detail.The optimum conditions were that enzyme conjugate was diluted to1:3000before use andthe PBS solution in pH7.02was selected as the preparation solution in the following experiments.（3） Biomimetic enzyme-linked immunosorbent assay （BELISA） method for the rapiddetermination of acrylamide. Under the optimal conditions, the established BELISA methodhad a good sensitivity (IC50,8.0±0.4mg L^-1) and a low limit of detection (IC15,85.0±4.2μgL^-1). The Cross-Reactivity （CR） of glycinamide hydrochloride, acrylicacid and propionamidewere all less than16%. The blank potato samples spiked with acrylamide at three levels of100,250and500μg L^-1were extracted and determined by the proposed method, and goodrecoveries ranging from90.0%to110.5%were obtained. The acrylamide in French fries andCracker samples was quantitatively detected by the BELISA method with different levels of0.440±0.016mg kg^-1and0.424±0.028mg kg^-1, respectively. Compared with GasChromatography, the applicability of this developed BELISA method was validated. Theresults obtained by these two methods were correlated well and had no significant difference（P>0.05）.