Research Of Simultaneous Detection Method Of Organophosphorus Pesticides Residues In Vegetables Based On Three-dimensional Aptasensors

Organophosphorus pesticides（OPs）are widely used in the production of agricultural products because of their efficacy in controlling crop diseases and insect pests.However,the residual OPs in agricultural products or the environment can enter the human body and seriously endanger the neurological function of the human body.Especially long-term low-dose exposure to OPs can lead to muscle dysfunction,tumorigenesis,and poor reproduction.Contaminated food caused by pesticide residues has become a global concern for food safety,health protection,and public safety issues.At the same time,the diversity and complexity of pesticide residues have brought great challenges to detection.The traditional analytical techniques for various OPs residues have high sensitivity and selectivity,but there are still some drawbacks,such as time consuming,complicated operation,and requiring expensive special equipment.Therefore,it is an effective way to solve this problem to develop a rapid,simple,and sensitive simultaneous detection strategy for multiple pesticide residues.In recent years,aptasensor has been widely used in the detection of OPs.Aptamer（APT）is an oligonucleotide sequence that has been screened in vitro and can bind to the corresponding target with high affinity and specificity.Compared with single-stranded DNA（ss DNA）,double-stranded DNA molecules are not easy to bend and fold,therefore,DNA tetrahedral nanostructures have been introduced into the construction of aptasensors in recent years.DNA tetrahedral nanostructure is a new type of DNA structure,which not only has a relatively stable three-dimensional rigid structure but also can reduce the local overcrowding effect on the electrode surface by constructing spatially separated overhanging probes,thus effectively improving the binding efficiency to the target substance and the resistance to interference of aptamer sensors.In this paper,aptamer was used as the recognition element,a more stable aptamer nanostructure was designed and constructed,and the construction method of the sensor for simultaneous detection of various organophosphorus pesticide residues was explored.The main research contents were as follows:（1）The aptamer sequence recognizing a variety of OPs was carried out targeted cutting and reconstruction,gold nanoparticles（Au NPs）and DNA tetrahedral nanostructures（DTN）were used to assemble a pyramid-shaped nucleic acid probe carrier（Au NPs-DTN）to construct an ultrasensitive electrochemical aptasensor（Au NPs-DTN sensor）.The designed Au NPs-DTN structure could not only reduce the steric hindrance but also improve the hybridization efficiency of aptamer and target material.In addition,the performance of the constructed sensor was compared with that of the sensor based on a single-stranded aptamer（ss APT）,and it was found that the experimentally designed Au NPs-DTN sensor had higher sensitivity.Under the optimal detection conditions,the detection limits of phoxim,fenitrothion,fenthion,parathion,and methyl parathion were 0.07 pg/m L,0.8pg/m L,0.7 pg/m L,0.7 pg/m L,and 0.08 pg/m L respectively.The developed Au NPs-DTN biosensor was applied to the real vegetable samples and phoxim recoveries ranged from91.6%to 106.6%（n=3）.（2）In order to realize the simultaneous detection of a variety of OPs residues,we developed a dual-ratiometric electrochemical aptasensor that could detect malathion（MAL）and profenofos（PRO）simultaneously.In this study,metal ions,hairpin-tetrahedral DNA nanostructures（HP-TDN）and nanocomposite materials were used as signal tracers,sensing framework and signal amplification strategy respectively to develop the aptasensor.Thionine（Thi）labeled with HP-TDN(HP-TDN_Thi)provided specific binding sites for assembling Pb²⁺labeled MAL aptamer(Pb²⁺-APT₁)and Cd²⁺labeled PRO aptamer(Cd²⁺-APT₂).When the target pesticides were present,Pb²⁺-APT₁ and Cd²⁺-APT₂dissociated from the hairpin complementary strand of HP-TDN_Thi,resulting in reduced oxidation currents of Pb²⁺(I_Pb²⁺)and Cd²⁺(I_Cd²⁺),respectively,while the oxidation currents of Thi(I_Thi)remained unchanged.Thus,I_Pb²⁺/I_Thi and I_Cd²⁺/I_Thi oxidation current ratios were used to quantify MAL and PRO,respectively.Meanwhile,the gold nanoparticles（Au NPs）encapsulated in the ZIF-8 nanocomposite（Au@ZIF-8）greatly increased the catch of HP-TDN,thereby amplifying the detection signal.The HP-TDN with a rigid three-dimensional structure could greatly improve the assembly and recognition efficiency of aptasensor,which makes the HP-TDN-based aptasensor have high sensitivity,reliability,and anti-interference ability.Under the optimal detection conditions,the detection limits of the HP-TDN aptasensor for MAL and PRO were 4.3 pg/m L and 13.3 pg/m L,respectively.