| Peanut allergy can cause gastrointestinal discomfort, allergic dermatitis and other allergicreactions. It evev can cause anaphylactic shock and anaphylactic death. At present, there areno effective treatments to peanut allergy at home and abroad. Therefore, to avoid sensitivegroups eating peanut-containing food is recognized as the most effective mean to preventpeanut allergy, currently. So the research to establish a rapid and sensitive method to detectpeanut allergen is of great significance. Compared to proteins, DNA can keep stable for a longtime under processing conditions, such as heating and pressure. So the residual peanutallergen DNA can better indicate the present state of allergens in food.The specific “molecular beacon”-liked stem-loop probe of main peanut allergen Ara h1and its complementary target sequence were synthetized in this article. And based on theprinciple of DNA hybridization, three stem-loop DNA electrochemical biosensors wereestablished.1. The stem-loop probe and6-thiolhexanol were self-assembled on the surface of thegold electrode to build a simple DNA biosensor without amplifying signal. Theelectrochemical impendance spectroscopy was used as the measuring method, which candetect target gene with high sensitivity. The detection linear range was from10-15mol/Lto10-10mol/L, and the detection limit was3.5×10-16mol/L. The method was also used in thedetection of Ara h1genes in peanut milk beverage with the concentration of （3.23±0.20）×10-13mol/L, the recoveries from72.9%100.2%, and relative standard deviations from5.1%7.9%.2. The graphene–gold nanocomposite was prepared by graphene and gold nanoparticleswere electro-deposited on the surface of the glassy carbon electrode. The DNA biosensor wasmodified by the nanocomposite and gold film, and differential pulse voltammetry was used asthe detection method with the linear range from1.0×10-16mol/L to1.0×10-13mol/L anddetection limit4.1×10-17mol/L. The method was also used in the recovery test of the peanutmilk beverage, with the recoveries from86.8%to110.4%, and relative standard deviationsfrom2.3%to6.7%. The method improved the recovery, sensitivity and specifity.3. The spongy gold film was immobilization on the glassy carbon electrode modified bychitosan-multi-walled carbon nanotube composites （CS-MWCNT）. The electrochemicalDNA biosensor was constructed by self-assembling stem-loop probe on the gold film. By thesignal amplification of horseradish peroxidase （HRP） catalysis, the redox signal of H2O2wasmeasured by amperometric current-time curve, which can detect target gene ultrasensitivelywith the linear range from3.91×10-17mol/L to1.25×10-15mol/L, and the detection limit1.3×10-17mol/L. The method was also used in the detection of Ara h1gene in peanuts with the concentration of （2.74±0.11）×10-11mol/L. The method was most sensitive, but with a bitpoor stability.The specificity of the above three methods were all good, and they were also reproducedand stable. Due to the amplification of the nano material and the enzyme modified on theelectrode surface, the sensivity of the three methods were gradually increased, but the linearrange gradually narrowed. In addition, the SYBR GreenⅠ real-time PCR was also establishedto verify the quantitative reliability of the DNA biosensors. The result showed that thequantitative detection of the electrochemical DNA biosensor was realiable.The stem-loop DNA biosensors established by this study can all used in the detection ofAra h1gene in food sample. The DNA biosensor has good prospects for development, andprovides the train of thought for food allergen detection. |
PDF Full Text Request