| IARC (International Agency for the Research of Cancer) classified acrylamide (AA) as "Group 2A" substance (probably carcinogenic to humans) in 1994 due to its neurotoxicity, potential carcinogenicity, and genotoxity. Swedish National Food Agency reported that a large amount of AA was found in starch-rich thermally processed foods in 2002. Since then, AA has drawn much greater attentions in the fields of food safety, toxicology, and analytical chemistry. Standard methods for the detection of AA have advantages of high sensitivity, accuracy, and repeatability, but require expensive instruments, skilled technicians in laboratories, and high test cost, which cannot meet the needs of rapid, in-field or on-line detection of AA. Optical chem/biosensors have been studied in analysis, diagnosis, and detection of biological and chemical targets due to their high sensitivity, good selectivity, low cost, and automated operation. For small molecules with toxicity or no immunogenicity, it is difficult to obtain their antibodies via immunoreaction. Aptamers, as "chemical antibody", which are obtained through in vitro selection, can make up the weakness of antibodies. In addition, the development of biosensors using aptamers as recognition elements would provide more opportunities in the future for the detection of AA.In this research, nanotechnology, fluorescent analysis, colorimetric analysis, quartz crystal microbalance (QCM), and systematic evolution of ligands by exponential enrichment (SELEX) have been used to establish optical chem/biosensors to achieve rapid, sensitive, and selective detection of AA.The three approaches with their results and conclusions are briefed as the follows:(1) Fluorescent chemical sensor for rapid detection of AAA novel fluorescent sensing method based on AA polymerization-induced distance increase between quantum dots (QDs) was proposed for detecting AA rapidly. The functional QDs were prepared first by conjugating with N-acryloxysuccinimide (NAS), which was characterized by Fourier transform infrared (FR-IR) spectra. The carbon-carbon double bonds of NAS modified QDs polymerized with assistance of photo initiator under UV irradiation, leading to QDs getting close along with fluorescence intensity decrease. In the presence of AA, they participated in the polymerization and induced an increase of the distance between QDs, resulting in the fluorescence intensity enhancement. At the optimized conditions, this method possessed a linear range from 0.5 to 5×104μmol L-1 with a limit of detection of 0.5μmol L-1. All compounds similar to AA in chemical structure did not generate detectable signals except L-asparagine. Therefore, L-asparaginase was used to eliminate the distractions from L-asparagine. Application of this method to detect AA in potato chips and cookies show satisfactory results compared with standard methods. Although the sensitivity is not as good as LC-MS/MS, this fluorescent sensing method also opens a new way to detect AA, which has great potential for rapid, quantitative, and on-line detection of AA in thermally processed foods.(2) Colorimetric biosensor for sensitive detection of AAA novel colorimetric method was proposed for the visual detection of AA based on a nucleophile-initiated thiol-ene Michael addition reaction. Gold nanoparticles (AuNPs) were aggregated by glutathione (GSH) because of a ligand-replacement, accompanied by a color change from red to purple. In the presence of AA, after the thiol-ene Michael addition reaction between GSH and AA with the catalysis of a nucleophile, the sulfhydryl group of GSH was consumed by AA, which hindered the subsequent ligand-replacement and the aggregation of AuNPs. Therefore, the concentration of AA could be determined by the visible color change and absorption spectra caused by dispersion/aggregation of AuNPs. This new method showed higher sensitivity with a linear range from 0.1 to 80μmol L-1 and a detection limit of 28.6 nmol L-1, and better selectivity than the fluorescent sensing method. Moreover, this new method was used to detect AA in potato chips with a satisfactory result in comparison with the standard methods, which indicated that the colorimetric method can be expanded for the rapid detection of AA in thermally processed foods.(3) Colorimetric aptasensor for selective detection of AA with selected aptamersAptamers, known as "chemical antibodies", are DNA or RNA sequences specific binding with targets. Optical aptasensors have been widely studied to analyze small molecules. To further improve specificity, a colorimetric aptasensor was designed to detect AA selectively. First, aptamers specific to AA were selected via a QCM-SELEX method. The selection started with a single-stranded DNA (ssDNA) library of 1015 molecules randomized at central 45 nt. During the selection, the binding affinity of ssDNA pool in each round can be real-timely characterized by the change of frequency (△f) before and after incubation with AA. After 14 rounds of selection and 4 rounds of counter selection, AA aptamer candidates were enriched. After cloning, sequencing, homologous analysis, and simulation of secondary structure, two aptamer (A5 and C14) candidates were selected and characterized by QCM and dot blot assay. The dissociation constants of A5 and C14 were 17.2 nmol L-1 and 115.2 nmol L-1, respectively. Both A5 and C14 didn’t show affinity to acrylic acid, methacrylic acid, and methacrylamide. Finally, a label-free colorimetric aptasensor was established to detect AA rapidly and selectively with selected aptamers. The linear range was from 0.5 to 10μmol L-1 with a detection limit of 0.39μmol L-1. |
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