Florescent And Electrochemical Aptasensor For Rapid Screening Of Antibiotic Residues In Foods

Antibiotics have played an important role in the treatment of human diseases since they were invented.However,due to the abuse of antibiotics,antibiotic residues have been found in the food and environment and can enter the human body through the food chain,affecting our health.The presence of antibiotics in foods and the like is rare;secondly,the composition of foods is complex,and the effects of detection and interference are severe;in addition,all kinds of antibiotics are generally present at the same time.Thus,it is important to develop rapid,high-sensitivity,highselectivity and simultaneous detection assays of trace amounts of antibiotics.In this paper,we have developed an enzyme-free and label-free aptasensors based on electrochemical and fluorescent methods to detect antibiotic residues(chloramphenicol,Kanamycin,oxytetracycline)in foods with high sensitivity and rapid detection.Combined with the stirring bars,the reaction rate is accelerated,phase separation is facilitated,and the background signal is reduced.And these can successfully achieve antibiotic residue detection in food,and show potential application value in food safety testing.This work is mainly carried out from the following three parts: 1.Enzyme-and label-free electrochemical aptasensor for Kanamycin detection based on double stir bar-assisted toehold-mediated strand displacement reaction for dual-signal amplificationIn this study,an enzyme-and label-free electrochemical aptasensor for antibiotics,with Kanamycin(Kana)as a typical analyte,was developed based on a double stir bar-assisted toeholdmediated strand displacement reaction(dSB-TMSDR)for dual-signal amplification.First,we modified two gold electrodes(E-1 and E-2)with different DNA probes(S1/S2 hybrid probe in E-1 and DNA fuel strand S3 in E-2).In the presence of Kana,an S1/S2 probe can be disassembled from E-1 to form an S2/Kana complex in supernatant.The S2/Kana could react with S3 on E-2 to form S2/S3 hybrid and release Kana through TMSDR.After then,the target recycling was triggered.Subsequently,the formed S2/S3 hybrid can also trigger a hybridization chain reaction(HCR).Consequently,the dual-signal amplification strategy was established,which resulted in many long dsDNA chains on E-2.The chains can associate with methylene blue(MB)as redox probes to produce a current response for the quantification of Kana.The assay exhibited high sensitivity and specificity with a detection limit at 16 fM Kana due to the dual-signal amplification.The double stir bars system can both increase phase separation and prevent leakage of DNA fuel to reduce background interference.Moreover,it allows flexible sequence design of the TMSDR probes.The assay was successfully employed to detect Kana residues in food and showed potential application value in food safety detection.2.Enzyme-free fluorometric assay for chloramphenicol based on double stirring bar-assisted dual signal amplificationAn enzyme-free fluorometric assay is described that accomplishes dual signal amplification by making use of a two stirring bars.Two Y-shaped DNA probes were designed and placed on the bars.When the target(with chloramphenicol as model analyte)is added,it triggers target recycling and simultaneously catalyzes hairpin assembly(CHA).A large fraction of DNA primers is released by the analyte from the bar to the supernatant and open hairpins with G-quadruplex DNA sequence.The G-quadruplex can specifically bind thioflavin T(ThT)to emit fluorescence(with excitation/emission maxima at 445 and 485 nm)for quantification of chloramphenicol.An enzyme is not needed.ThT is added to the system as a fluorescent DNA probe.All this strongly reduces the cost for sensor construction and usage.The dual signal amplification steps occur simultaneously which reduces the detection time.The assay was successfully employed to the determination of CAP in spiked milk and fish samples within 60 min and with a 16 pM limit of detection(at S/N = 3).3.A novel electrochemical aptasensor for simultaneous detection of three tumor markers using MOF labeled DNA scaffold encoded probesWe have developed an enzyme-and label-free electrochemical aptasensor for multiple antibiotic detection(Kana and OTC as typical analytes),which a dual-enriched signal source is based on MOF-labeled DNA scaffold coding probes.First,we used hierarchically porous UiO-66(HPUiO-66)to adsorb methylene blue(MB)and ferrocene(Fc)for signal tags.The phosphate-modified DNA(P-DNA)is then immobilized on the MOF to which the signal source is adsorbed by forming a Zr-phosphate bond,and hybridized with the HCR product on the gold nanoparticle-modified gold stirring bars to further enrich the MOF to achieve dual signal source enrichment.The dual enrichment of DNA and MOF to signal substances enables high sensitivity for simultaneous antibiotic detection.Secondly,the modification of DNA to MOF with Zr-phosphate bond avoids complex coupling processes and improves experimental efficiency.Finally,the detection time is greatly reduced by preparing the signal probe in advance without participating in the signal amplification strategy in the actual detection process.In addition,the proposed dual-enriched signal source measurement can be combined with related signal amplification strategies to further increase sensitivity.The test was successfully used to detect Kana and OTC residues in foods and showed potential application value in food safety testing.