Study On The Determination Of Chloramphenicol In Milk Based On DNA Templated Silver Nanoclusters

Long-term consumption of food containing antibiotic residues may lead to human suffering from many diseases,such as cardiovascular,enteritis and gastritis,liver and kidney function damage,anaphylactic reaction and so on.At present,chloramphenicol(CAP)is one of the most common residual antibiotics.Recent reports show that the detection rate of chloramphenicol in animal derived food is very high.Therefore,it is necessary to develop rapid and sensitive detection methods to enhance the monitoring of chloramphenicol residues.DNA-template silver nanoclusters(DNA-AgNCs)have the advantages of simple synthesis,tunable wavelength,and low preparation cost.Various sensors based on DNA-AgNCs have the characteristics of multi-color detection and flexible signal output,which have attracted extensive attention of researchers in recent years.They applied to chemical analysis,environmental detection,food safety,disease diagnosis,medical treatment,and other fields.In this dissertation,CAP was taken as the detection object,the identification probe was designed based on CAP aptamer,and DNA-AgNCs were used as the signal output mode of the probe to design three fluorescence detection schemes for CAP,the contents are as follows:(1)An"on-off"fluorescence detection method based on CAP aptamer self-template was constructed.Based on the principle of CAP aptamer hairpin formation mediated by C-Ag-C,the DNA-AgNCs synthesis conditions were studied.After optimization of the concentration ratio of silver nitrate to DNA,buffer type,p H,reaction time,and other synthesis conditions,the method can achieve rapid detection of CAP at room temperature,and obtain DNA-AgNCs with a quantum yield of 16.36%.Then,based on the quenching response of CAP to the DNA-AgNCs,a fluorescence method for the detection of CAP was established.Besides,the fluorescence signal showed a clear linear dependence(R~2=0.997)on the CAP concentration over the range from 0.5 to 20 nmol/L.The regression equation was Y=0.02867X+0.15709 with the limit of detection(LOD)estimated to be 0.052 nmol/L(3 times the signal-to-noise ratio calculation).(2)An"off-on"CAP detection method based on DNA three-way junction was established by using Toehold-mediated Strand Displacement Reaction Technology.The probe design of three-way junction was carried out,including the complementarily number of aptamer bases,the arm length of three-way junction base,and the template sequence of AgNCs,etc.DNA-AgNCs with good fluorescence quantum yield(17.54%)were synthesized and characterized by TEM.After optimization of the experimental conditions such as buffer solution,p H,reaction time,etc.,the"off-on"fluorescence detection method of CAP was established.Besides,the fluorescence signal showed a clear linear dependence(R~2=0.9981)on the CAP concentration over the range from 1 to10 nmol/L.The regression equation was Y=0.0484X+0.8523 with the limit of detection(LOD)estimated to be 0.89 nmol/L(3 times the signal-to-noise ratio calculation).(3)A two-signal ratio detection method based on dimer AgNCs and DNA three-way junction was developed.Firstly,DNA-AgNCs with red and yellow fluorescence emission were obtained through a series of synthesis and screening of DNA templates,and were characterized by TEM.Secondly,the number of base connections of the three-way junction probe was optimized by electrophoresis and fluorescence experiments,and then after optimization of the reaction conditions,such as the concentration ratio of silver nitrate,sodium borohydride and DNA,p H,buffer solution,temperature,synthesis parameters of AgNCs.Finally,the fluorescence signal showed a clear linear dependence(R~2=0.9887)on the CAP concentration over the range from 0.01 to 0.5 nmol/L.The regression equation was Y=1.0543X+0.0867 with the limit of detection(LOD)estimated to be 9.8pmol/L(3 times the signal-to-noise ratio calculation).