The Development Of Immunoassays For The Detection Of Acrylamide Produced In Fried Potato Chips

Fried potato chips are crispy and have a variety of tastes,which are more and more popular among people,especially for teenagers and children.However,these starch-rich fried foods tend to produce small molecular substances such as acrylamide during processing,and their residual increases with increasing temperature.Studies have shown that acrylamide is carcinogenic,genotoxic,neurological and reproductive developmental toxicity,and long-term intake can affect the health of the human body.In this paper,we developed immunoassays for rapid,sensitive and high throughput detection of acrylamide produced in fried potato chips,which are using HRP and C-Dots as labels,respectively.The main research contents are as follows:（1）Three kinds of acrylamide haptens were synthesized and characterized.The anti-acrylamide polyclonal antibodies were prepared by animal immunization with three haptens binding to carrier protein as immunogens.By indirect enzyme-linked immunosorbent assay,the obtained antisera were determined under the conditions of homologous and heterologous assays.The antiserum which used AA-4-MBA-BSA as the immunogen had the highest titer and inhibition rate under the condition of homologous coating antigen.The antiserum was also purified to obtain the anti-acrylamide antibody for the next experiments.（2）The enzyme-linked immunosorbent assay was developed.The optimal antibody dilution and coating antigen concentrations were obtained by checkerboard titration.The conditions were tested by optimized experiment.The coating antigen was at 4℃for 12 h by using homologous coating antigen.The optimal blocking and antigen dilution buffer were 1%BSA/PBS and PBS（pH=7.4）containing 10%methanol,respectively.The competition time was 45 min.Under the reaction conditions,the acrylamide hapten was detected by indirect competitive enzyme-linked immunosorbent assay,The sensitivity(IC₅₀)was 55μg/L and the limit of detection(IC₁₅)was 2μg/L.The recoveries of spiked potato chips were 73%⁸6%,and the results were also validated by HPLC with good consistency.The assay could be applied for the detection of acrylamide effectively in potato chips.（3）The fluorescence immunoassay was also developed:Carbon quantum dots were synthesized by hydrothermal method,and then coupled with anti-acrylamide antibodies to form fluorescent probes.The bio-conjugate were characterized by ultraviolet spectral scanning,fluorescence scanning,and gel electrophoresis,and developed fluorescence immunoassay for the detection of acrylamide.The standard curve of the assay showed a good linear relationship when the concentration of acrylamide hapten was 0.32¹000μg/L.The recoveries of acrylamide in Fried potato chips was 76%⁸9%with good consistency validated by HPLC.Compared with the established enzyme-linked immunoassay method,it possessed the advantages of simplicity,convenience and shorter analysis time without the addition of substrate.