A Novel Detection Method Of Genetically Modified Crops Based On Biosensor

With the commercialized planting of genetically modified crops around world,governments and the public are paying more attention to the safety of genetically modified organisms?GMO?.In order to strengthen the supervision of genetically modified crops and protect consumers'right to know,development of novel technology has been a challenge.Therefore,it is necessary to establish a set of rapid,sensitive and accurate methods for transgenic detection.At present,the classical and standardized methods for the detection of genetically modified crops are based on the nucleic acid,such as qualitative PCR real-time PCR and digital PCR.The advantages of these methods are that most of the laboratories are capable to meet the conditions of equipments.However,the disadvantages are that they take a long time for amplification,and all the regents and accessories cannot be regenerated.Therefore,sensor chips that are rapid,sensitive,lable-free and reproducible have attracted researchers'attention gradually.Surface plasmon resonance?SPR?biosensor is a type of optical sensing system,using the optical angle shift to achieve accurate detection of various analytes.It not only guarantees the specificity and sensitivity,but also has the advantages of label-free,real-time,regeneration and calibration free of concentration analysis.Therefore,it has become a focus in recent years.In this dissertation,t-nos,CaMV35S,and cry1A were selected as analytes,the qualitative and quantitative detection methods for transgenic crops based on SPR were established.The key parameters of the methodology,such as specificity,sensitivity and kinetic characteristics,were also investigated.The results showed that:?1?The SPR biosensor constructed for qualitative detection could simultaneously analyze the elements of t-nos,CaMV35S and cry1A,with good specificity and sensitivity.Immobilization level of probes on the surface can affect the detection sensitivity.The limit of detection?LOD?was becoming lower with the increasing of immobilization levels.However,in the same level of immobilization,high?G+C?%content and simple secondary structure would decrease the LOD,which was LOD_?t-nos?<LOD_?CaMV35s?=LOD_?cry1A?.For detection of multiple targets,it was found that the sensitivity of the same target in the form of single substance and mixture were different,and the sensitivity of the target in mixture was poor,indicating that other components in the mixture could also affect the sensitivity.Random cry1A probe was used in different species,rice,corn,soybean were chose,the affinities of three kinds of target DNA with this random probe were k_D?rice?=2.32×10^-9 M,k_D?corn?=3.10×10^-9 M,k _D?soybean?=4.17×10^-9 M,respectively.The results suggest that affinities between random probe designed and three kinds of cry1A target were very high and had no significant difference.Therefore,the random cry1A probe can be used to screen several cry1A insect-resistant crops with good sensitivity and specificity.?2?The calibration free concentration analysis?CFCA?method based on the SPR principle can achieve the quantitation of t-nos samples when the calibrator was absent.The within-day precisions were from 1.61%to 2.10%,and between-day precision was 1.78%,which implied that CFCA method has good repeatability and reproducibility performance.Before CFCA assay,kinetics were investigated,association constant?k_a?was more than 5×10⁴ M^-1s^-1,and equilibrium constant?k_D?was less than 10^-6 M of t-nos target and its probe.It implied that both association constant?k_a?and equilibrium constant?k_D?met the preconditions CFCA assay.For optimizing CFCA method,when the immobilization levels ranged from 1400 to2000RU,QCratio was greater than 0.2,and the relative standard deviation?RSD?of the concentration analysis in different dilution levels were below 5%,indicating that results of quantitation by this method were accurate and reliable.Then the same sample was analyzed with standard method,the results of one-way ANOVA showed that no significant difference occurred between two methods within the 95%confidence interval.The SPR biosensor constructed in this dissertation can achieve the qualitative and quantitative detection of a variety of transgenic DNA analytes,with the advantages of rapidity,specificity and sensitivity,independent of the standard curve for concentration analysis,and the chip can be regenerated at the end of each cycle.Therefore,it is expected to achieve the detection of DNA analytes with high throughput,large-scale and good economic benefits.