Construction Of Aptamer Microarray For Broad Spectrum Organophosphorus Pesticide Detection

With China’s national economy continuing to grow and people’s living standards improving,people pay more attention to the food safety on the table.Among them,organophosphorus pesticides(OPs)residues are still a serious threat to the physical and mental health of people.As a result,it is necessary to strengethen supervision and develop OPs detection techlogy in order to protect the people’s last ‘line of defense’.However,there are still a few drawbacks in these detection technologies,such as cumbersome operation,expensive instruments and difficult onsite detection.It is therefore particularly important to develop novel and efficient detection methods to suit different detection needs.An aptamer microarray method for high-throughput and rapid detection of OPs was successfully developed based on the principle that OPs and a fluorescent dye Thioflavin T(Th T)compete to combine with the aptamer of Gquadruplex conformation,resulting in a change in the system’s fluorescence signal.The specific work of this research is as follows:1.Before constructing the aptamer microarray,the feasibility of reaction principle was preliminarily investigated in solution by fluorescence method and UV method.The experimental results show that the reaction principle is consistent with the assumption and can be used for the detection of OPs,i.e.,the free Th T in solution has essentially little fluorescence signal.After binding with the aptamer,the G-quadruplex conformation of the aptamer activated the potential fluorescence properties of Th T.At this time,the fluorescence signal of the system increased significantly and reached the maximum.After adding OP3,partial aptamer preferred to bing to OP3,resulting in the dissociation of Th T from the G-quadruplex,and the fluorescence signal of the system decreased.Based on this strategy,10 to 200 μM OP3 were successfully detected under optimized conditions,which determined the appropriate target concentration for subsequent microarray experiment.2.Through the Schiff base reaction between the aptamer terminal amino group and the aldehyde group on the chip surface,the reaction in solution was successfully transferred to the aptamer microarray platform.Under the optimized conditions(300 n M PES11-12 T,80 μM Th T),the developed approach was used to detect OP3 firstly and the limit of detection was calculated to be 0.52 μM according to the 3σ rule.Then eight OPs except OP3 were detected to investigate the broad spectrum of the aptamer microarray method,and the OPs with high fluorescence inhibition rates were quantified.Spiked recovery experiments in pear and radish extracts were used to assess the method’s applicability and accuracy,and the developed method has obvious advantages over other methods in terms of low-cost high throughput and rapid detection.3.As the principle of this research involves G-quadruplex conformations,the possible reaction mechanism in 1× binding buffer was explored based on circular dichroism spectrum.The effect of the four cations in the buffer composition on the formation of G-quadruplex and the possible reaction mechanism of PES11 in Tris-HCl buffer without any cations were then verified,and fluorescence experiments and PES11 truncation experiments revealed the formation of intramolecular parallel G-quadruplex by PES11 in Tris-HCl buffer.Finally,the fluorescence inhibition rate of the PES11-Th T complex by OP3 was investigated in both buffers,and it was determined that 1×binding buffer was more suitable for this system by comparison.