Fluorescent Sensor Assay For β-lactamase In Milk Based On A Combination Of Aptamer And Graphene Oxide

β-lactamase, a kind of inactivated enzyme, can be used to hydrolyze penicillin and cephalosporin antibiotics. In order to achieve the aim of masking of antibiotics residue or "no anti-milk" production, so the enzyme was illegally added to food or food raw materials (such as milk and raw milk) by criminals. If people continually consumer milk products with illegally added β-lactamase for a long time, then bacterial resistance will be increased in the human body. As a result, "super-bacteria" will be produced, moreover allergic nettle rash, fever, and even anaphylactic shock hazard can be caused; on the other hand, after the decomposition of antibiotics in β-lactamase, other new harmful substances may be produced, such as penicilloic acid. Therefore, it is very important to establish an efficient, reliable and sensitive fluorescent sensor assay (FSA) to detect rapidly the residue of β-lactamase in milk products, thus helping to fight against illegal and criminal acts in the consumer product field, ensuring food safety and human health.In this paper, natural flake graphite powder was prepared of graphene oxide (GO) by the Hummers method, using a UV-vis spectrophotometer (UV-vis), Infrared spectrometer (IR), X-ray diffraction (XRD), Raman spectrometer (Raman), and Thermal gravimetric analysis (TGA) to identify the GO, and additionally its appearance was analyzed by Electron microscopy. Carboxyfluorescein (FAM) modified P-lactamase aptamer (FDNA) was then used to replace the traditional antibody to bind with β-lactamase. A process by which was identified by agarose gel electrophoresis and UV-vis spectrophotometer. The principle of fluorescent sensor assay established in this study is that, without β-lactamase, FDNA will adsorb on the surface of GO by π-π conjugated interaction, with fluorescence resonance energy transfer (FRET) between GO and FDNA, GO can quench the fluorescence signal of FDNA so that the system is in low fluorescence signal. On the other hand, when the system in the presence of β-lactamase, FDNA will bind to β-lactamase and hinder the fluorescence energy transfer between FDNA and GO, thus releasing a high fluorescence signal. After optimizing the experimental principle and the FDNA verification and the experimental conditions, based on the FDNA and GO, FSA was established to detect the β-lactamase in milk products, with a detection limit of 0.5 U/mL, the linear range of 1-46 U/mL, the correlation coefficient R2=0.999. Compared with the ELISA method, FSA showed reliability and a high correlation value of 0.993 with the ELISA results. For the recoveries, the spiked recoveries range from 96.04%-119.67% by FSA with less than 6.70% for each sample while it ranged from 93.10%-119.06% by ELISA with less than 7.93% for each sample. The experimental results indicated that the fluorescent sensor assay established in this study is reliable, sensitive and highly specific for the analysis of milk sample for β-lactamase residual.