Reaserch Of Aminoglycoside Antibiotics Residues Detection Method Based On Aptasensor And Its Application In Milk

Aminoglycoside antibiotics(AGs)are widely used in animal husbandry because of their advantages of high efficiency and low price.However,the abuse or irregular use of AGs has caused the problem that excessive residues in animal foods such as milk become more and more serious,which would seriously endanger public health.At present,the main detection methods for AGs residue still rely on large instrument analysis method with expensive equipment,large manpower and material consumption.Although their accuracy is high,they cannot meet the requirement of rapid on-site detection.Therefore,the development of efficient and sensitive AGs residue detection methods has become the main research focus.As the cognitive components of biosensors,aptamers have the advantages of convenient modification,adaptive combination and good stability.Sensor analysis methods based on specific aptamers could solve the problems of different types of AGs detection.In this paper,electrochemical and fluorescent methods,which were widely used in biosensors,were used to construct two kinds of aptasensors by combining specific aptamers with nanomaterials.And the two aptasensors were used to detect streptomycin(STR)and kanamycin(KAN)residues that were commonly found in milk samples.The main contents were as follows:(1)A dual-amplification electrochemical aptasensor was constructed based on a screen-printed carbon electrode(SPCE)modified with graphitized multi-walled carbon nanotubes(MWCNT_Gr)and Au nanoshells as the test platform,and the aptasensor was successfully applied to the detection of STR.MWCNT_Gr had excellent electrical conductivity and Au nanoshells had a large specific surface area.The combination of these two could greatly promote the rate of electron transfer on the electrode surface and realize the double amplification of current signal.At the same time,it can provide more binding sites for STR aptamer,which 5'-terminally was modified amino,then realizing the sensitive detection of STR.Under the optimized conditions,the limit of detection(LOD)of the aptasensor for STR could reach 0.069 n M(S/N=3).(2)A hairpin aptamer(HP)was designed by using KAN aptamer.Its 5'end was modified with sulfhydryl group(-SH)to connect gold nanoparticles(Au NPs),and its 3'end was modified with amino group(-NH₂)to connect carbon quantum dots(CQDs).The Au NPs@HP@CQDs fluorescence aptasensor was successfully prepared for the detection of KAN.The fluorescence emission spectra of CQDs and the absorption spectra of Au NPs were overlapped.In the absence of KAN,CQDs and Au NPs would be close to each other due to the structure characteristic of HP,then the fluorescence signal of CQDs would be quenched by Au NPs due to occurence of the fluorescence resonance energy transfer(FRET).After KAN added,it could bind specifically to the aptamer and changed the structure of the aptamer,resulting in a gradual increase of the distance between CQDs and Au NPs.Then the FRET was broken and the fluorescence intensity would gradual recover.The recovery of fluorescence intensity could be characterized by fluorescence response and then used for the detection of KAN.Under the optimized conditions,the fluorescence intensity and logarithm value of KAN concentration was linear in the range of 0.01?500 n M(R²=0.9956),and LOD was 5.7 p M.(3)Streptomycin and kanamycin,the two kinds of common antibiotics in aminoglycoside antibiotics,were selected as targets.And the constructed electrochemical aptasensor and fluorescent aptasensor were successfully applied to the detection of AGs in milk samples.Taking into account that the fat in the milk composition might affect the signal conversion,the selected milk samples were treated with skimmed and non-skimmed pre-processing methods.The final results showed that the recovery rates of electrochemical detection of streptomycin were 94%?108%(non-skimmed)and 96.4%?103.8%(skimmed),and the recovery rates of fluorescence detection of kanamycin were 91.63%?99.80%(non-skimmed)and97.12%?105.20%(skimmed)respectively.The presence or absence of fat had a greater impact on the fluorescence method,and had little effect on electrochemistry.