Detection Of Kanamycin And Chloramphenicol By Fluorescent Aptamer Probe

The increasingly serious antibiotic abuse and misuse led to increased possibility of antibiotic residue in milk,livestock,fish and other foods with the widespread use of antibiotics.Traditional detection methods were time-consuming and expensive,such as microbial detection,instrument analysis,biosensor and so on.Therefore,it is of great significance to establish a fast and sensitive detection method.Aptamer is widely used in biological detection because of the characteristics of easy synthetic modification,good specificity and high affinity,Moreover,the fluorescent aptamer probes have the advantages of easy design and high sensitivity.In this study,two different fluorescent aptamer probes were designed for the detection of kanamycin(Kana)and chloramphenicol(CAP)in milk,respectively.The main findings are as follows:1.A fluorescent labeled aptamer probe was designed to detect Kana.Hairpin sequences labeled with FAM can complement with Kana aptamer to form hairpin conformation.Kana can specific binded with Kana aptamer which can dissociate from hairpin sequences.The free hairpin sequence labeled with FAM complementary to the sequence labeled with BHQ₂,resulting in fluorescence quenching.Based on the variation of fluorescence intensity before and after adding Kana,the quantitative determination of Kana was achieved.Under the optimal conditions,a good linear relationship was obtained in the ranged of 5.83?5.83×10³?g/L.The detection limit was 3.49?g/L with the response time of 15 min.Compared with the singly labeled aptamer probe,the sensitivity was significantly improved.This method has great potential in the field of food detection with the advantages of low cost,high efficiency and simplicity and so on.2.Fluorescent groups labeled on the aptamer can affect the combination of aptamer and target molecular,leading to reduced affinity.In order to avoid this problem,label-free aptamer probe was designed based on catalyzed hairpin assembly(CHA)and enzyme-catalyzed reaction.The detection method showed high sensitivity and selectivity using SGI dye(SYBR Green I,SGI)to detect CAP.Aptamer and primer formed double strand DNA complex.When the CAP was present,the double strand DNA complex dissociated,causing the primer to trigger four hairpins to form a continues four-arm junctions.After that T7 exonuclease(T7 Exo)was added to eliminate the unreacted hairpins.The four-way junctions were stained with SGI to produce a strong fluorescence signal.After a series of optimization experiments,a good linear relationship was obtained in the range of 0.001?10?g/L with detection limit of 0.72 ng/L.This developing method achieved signal cycle amplification without amplification equipment.Meanwhile,the addition of T7 Exo reduced the background signal interference,improved the detection sensitivity,and provided a new reference for food detection and biosensors.