Development Of A Fluorescent Immunochromatographic Strip Test Using Quantum Dot Submicrobeads For Chloramphenicol Detection

Chloramphenicol(CAP)is a kind of highly effective,broad-spectrum antibiotics widely used in animal husbandry and food processing industry for its charactoristics of low price,inhibitory effect.However there are some obvious side effects for CAP.These side effects include inhibition of bone marrow hematopoietic function,reproductive toxicity,neurotoxicity and so on.In order to protect the human health,most of the countries,including China,have set up laws to prohibit CAP usage for food-producing animals.However,CAP is still abused throughout the world.Therefore it is urgent to establish a rapid,convenient and specific method for CAP detection.Quantum dots(QDs)as a new type of fluorescent semiconductor nanomaterials exhibist many excellent optical properties,which has been widely used in various fields of analytical testing.Quantum dot submicrobeads(QBs)is assemble of a large number of quantum dots,which shows higher fluorescence intensity and better optical stability than quantum dots.In this paper,a fluorescent immunochromatographic strip test(ICST)based on QBs has been developed and applied to detect CAP in milk samples,the contents are as follows,1.Synthesis and indentification of complete antigen of CAPIn this work,CAP was conjugated with the bovine serum albumin(BSA)and ovalbumin(OVA)by carbonyl diimidazole method and carbodiimide method,then the complete antigens CAP-BSA,CAP-OVA,CAP-HS-BSA and CAP-HS-BSA were confirmed by ultraviolet spectroscopy and SDS-PAGE electrophoresis.The concentrations of complete immunogens confirmed by bicinchoninic acid(BCA)method were as follows,CAP-BSA was 6.8 mg/mL,CAP-OVA was 8.12 mg/mL,CAP-HS-BSA was 7,5 mg/mL and CAP-HS-OVA was 0.65mg/mL.The coupling ratio was 5:1 for CAP-BSA,2:1 for CAP-OVA,13:1 for CAP-HS-BSA and 19:1 for CAP-HS-OVA.2.The preparation of Quantum Dot Submicrobeads ProbeBy optimizing the ratio of coupling agent was 1:1,the pH value was 7.4,the ratio of quantum dots submicrobeads to antibody was 7.5:1,CdTe/ZnSe QBs-CAP McAb probes were successfully synthesized by conjugating QBs and anti-CAP monoclonal antibodies.The optimum storage temperature of the probe is 4? and the optimum dilution is 1.5%PVA+PBS(0.01mol/LpH7.4).3.Assembly and quality immunochromatographic strip test(ICST)A fluorescent immunochromatographic strip test(ICST)based on the quantum dot submicrobeads(QBs)has been developed for quantitative detection of Chloramphenicol(CAP).In this method,monoclonal antibody of CAP fluorescent probe was first prepared using the carbodiimide approach,then the concentration of 0.6 mg/mL for complete antigen CAP-HS-BSA was coated on nitrocellulose membrane as test line(T line).Similarly the concentration of 0.4 mg/mL for goat anti-mouse 1gG antibody was coated as control line(C line).The time required for the analysis was 15 min,and the limit of detection(LOD)for CAP was 0.1?g/L,with an analytical working range of 0.1-100?g/L.In spiking milk samples,the ICST demonstrated high recoveries in the range from 93.3%to 112.3%with low relative standard deviations from 3.1%-6.87%.