The Study Of Aptasensors For The Detection Of Ochratoxin A Based On DNA Self-assembly

Ochratoxin A(OTA)is a seriously toxic and carcinogenic fungal toxin with hepatotoxicity,teratogenesis,renal toxicity and immunotoxicity,and can dramatically damage human bodies.However,the characteristics of trace amount,highly toxic and the complex contaminated substrates for OTA dramatically enhance its determination difficulty.Traditional detection method with the drawbacks of complex process,high cost,low sensitivity,and poor repeatability and selectivity,etc,couldn't satisfy the requirements of micro-amount and multitudinous samples detection.So,a novel,economical,fast and handy detection method is developed critically with the characteristic of high accuracy and sensitivity.In this thesis,combined with technologies of DNA self-assembly,nano-palladium-minic enzyme,and magnetic beads,the electrochemical biosensor,optical biosensor and fluorescent biosensor were built and applied to the ultra-sensitive detections of OTA in different materials,respectively.1.An electrochemical biosensor on basis of DNA super-tree structures was developed to conduct ultra-sensitive detection of OTA in wheat,through the noulinear hybrid chain reaction,separation of magnetic beads and glassy carbon electrode determination.The magnetic sensor interface was formed firstly under the condition that capture probes,its part complementary OTA aptamer probe,and DNA were together added into magnetic beads,which was used to analysis,isolation and determination.And then under the existence of OTA,template probes and auxiliary probes,the DNA super-tree with biotin tags was formed on the surface of magnetic beads through aptamer binding reaction and series of nonlinear hybrid chain reactions.The super-tree was marked by the plentiful HRP with addition of the avidin-labeled HRP,and then catalyzed the oxidation of TMB by H2O2,accompanying with the occurrence of strong electrochemical signals.On absence of OTA,the super-tree was not formed,because the promoter DNA region on capture probes was blocked,so the nonlinear hybrid chain reaction wasn't triggered.The experiment results indicated that the sensor showed the good linear relationship in the range of 0.1?1 fg/mL under the optimum condition,the fitting formula was ?I=10.96C+4.92(R2=0.9925)and its limit of detection was 0.02 fg/mL.In the recovery test of OTA in wheat,the average recovery ratio was 99.3%.The results suggested that the sensor could recognize the OTA in complex system and was expected to detect practical samples.2.Based on DNA nanometer super-tree,an optical-visualization biosensor was built through Pd nano-particles mimic hydrogen peroxide and magnetic separation,and used to detect OTA in red wine.Briefly,the capture probes were assembled on the surface of magnetic beads and hybridized with its complementary OTA aptamer probes to form the double-strand DNA.On the existence of OTA,OTA was combined specifically to aptamer sequence and the promoter DNA fragments were exposed.Once addition of template and auxiliary probes,the nonlinear hybrid chain reactions were triggered and the DNA nanometer super-tree with mercapto modifications was formed on surface of magnetic beads.Subsequently the nano-Pd was coupled to the super tree.Because the nano-Pd possessed the catalase activity,it could catalyze the oxidation of TMB by H2O2.The color of reaction solution was become blue from colorless,and the blue solution possessed the UV-absorbance peaks at 371 nm and 653 nm.The absorption of reaction solution was detected at the maximum absorption wavelength of 653 nm using a UV spectrometry.The experiment results indicated that the sensor showed the good linear relationship in the range of 0.01?1.25 pg/mL under the optimum condition,the fitting formula was AA=0.757C+0.112(R2=0.9931)and its limit of detection was 6.1 fg/mL.In the recovery test of OTA in red wine,the average recovery ratio was 98.9%.The results suggested that the sensor possessed high-sensitivity and specificity and could was applied to detect OTA in red wine.3.On basis of DNA super.tree structures,a novel fluorescent biosensor was developed and used to rapid detect OTA in cocoa powder.Briefly,the capture probes were assembled on the surface of magnetic beads and hybridized with OTA aptamer probes.On the existence of OTA,the nonlinear hybrid chain reaction was triggered and the FAM-labeled DNA super-tree was constructed onto the surface of magnetic beads.The fluorescent intensity was detected with fluorescence spectrophotometer.The experiment results indicated that the sensor showed the good linear relationship in the range of 0.01?1 pg/mL under the optimum eondition,the fitting formula was AF=267.77C+34.56(R2=0.9979)and its limit of detection was 1.28 fg/mL.In the recovery test of OTA in cocoa powder,the average recovery ratio was 95.6%.The sensor with simple,rapid and inexpensive characteristics possessed a promising utilization potentiality in OTA detection.