Colorimetric Method Based On Aptamer And Hemin For Detection Of Chloramphenicol And Ochratoxin A In Foods

Chloramphenicol?CAP?is a highly effective broad-spectrum antibiotic that is often illegally used in livestock industry,which will cause food safety problems.Especially,it will residue in milk when used CAP in the lactation period of dairy cows.The residue of CAP has many effects for human health,such as hematopoietic function teratogenic and carcinogenic.Ochratoxin A?OTA?is a mycotoxin widely found in cereals,beans and fruits.If food is contaminated by OTA,long-term consumption of contaminated food can accumulate in the body and harm human health.At present,the methods for CAP and OTA detection have some disadvantages.For example,they require the large-scale instruments,expensive equipment,cumbersome operations,and complicated pre-processing.This thesis established colorimetric analysis detection methods for detection of CAP and OTA based on the specific recognition performance of aptamer and the mimic enzyme of hemin.These methods can be used for the rapid,sensitively and accurately detection of CAP and OTA in foods.This thesis includes the following two parts:Section 1,a colorimetric detection method was established for CAP detection in milk based on the specific recognition performance of aptamer and the mimic enzyme of hemin.Hemin were modified at 3'end of CAP aptamer and the 5'end of complementary strand,respectively.In the absence of CAP,two single-stranded DNA hybridized to form double-stranded DNA.Two hemin monomers formed hemin dimers which reduced their peroxidase activities and was thus unable to catalyze the oxidization of 3,3',5,5'-tetramethylbenzidine?TMB?by H₂O₂ to produce a blue colored product.In the presence of CAP,the CAP specifically hybridized with the aptamer.The DNA double strands were dissociated and the peroxidase activity of hemin monomers was restored which effectively catalyzed the oxidization of TMB by H₂O₂ to present blue colored product.The absorbance at 452 nm was recorded after adding of the stop solution.The optimization conditions were HAc-NaAc buffer?pH 4.0,100 mmol L^-11 Na⁺?,0.2?mol L^-11 DNA,0.9 mmol L^-11 TMB,and 100 mmol L^-11 H₂O₂,respectively.The colorimetric method showed a good linear response for CAP detection in the range of 1³0 nmol L^-1.The detection limit?3S/N?was 0.315 nmol L^-1.The recovery tested by the spiked CAP in milk samples was 95.5%¹16%,showing that the method can be used to sensitively,quickly and accurately detect CAP in milk,and the method has great potential for the detection of CAP residues.Section 2,a colorimetric method based on aptamers and hemin-G-quadruplex was developed for detection of ochratoxin A?OTA?in food.Two label-free single-stranded DNA?ssDNA?were designed.The ssDNA1 was the strand consisted of OTA-aptamer and a G-enriched sequence.The ssDNA2 was the complementary strand of the OTA-aptamer.In the absence of OTA,the two-ssDNA hybridized to form a double-stranded DNA.Hemin exhibited low peroxidase activity due to its aggregation in aqueous solution.However,in the presence of OTA,OTA specifically bound with the aptamer.The double-stranded DNA was damaged.Hemin bound with the G-enriched sequence and formed a hemin-G-quadruplex complex.This complex showed strong peroxidase-like activity and catalyzed the oxidation of hydrogen peroxide and 2,2'-azino-bis?3-ethylbenzothiazoline-6-sulfonic acid??ABTS?,the color of the solution changed from light green to dark green with a maximum absorption at 420 nm.The optimized conditions were PBS?pH 7.0?,1?mol L^-11 hemin,2 mmol L^-11 H₂O₂ and 4 mmol L^-11 ABTS,respectively.The colorimetric method showed a good linear response for OTA detection in the range of 0.001².5?mol L^-1.The regression equations were y₁=4.567x+0.312 and y₂=0.111x+0.356,respectively.The detection limit was 70.3 pmol L^-1?3S/N,n=10?.This method was applied to the detection of OTA in 10 kinds of foods such as peanuts,walnuts and red beans.It was found that the OTA content of noodles,breads,red beans,peanuts and chicken feed exceeded the national standard limit(5.0?g kg^-1=0.012?mol L^-1).The OTA spike recovery test was conducted in walnuts and peanuts,and the recovery rate was between 91.9%and 109%,indicating that the established method can accurately and sensitively detect the OTA content in food.It is significant to food safety.