Study On The On-site And In-situ Biosensing Methods For Organophosphorus Pesticide Residues On Crop Surfaces

Pesticides are necessary compounds in agricultural production,which play an essential role in the harvest of crops.Although they have superior effects on the prevention and control of pests and diseases,their inherent or potential toxicity and residue problem for animals and the environment are inevitable.With the development of research,people have realized the residue problems caused by the usage of some organophosphorus pesticides(OPs),such as dichlorodiphenyl trichloroethane(DDT),which even have caused a huge disaster.Consequently,it is of great importance to establish simple,rapid,and reliable methods for pesticide detection.Aimed at the significant demand of the on-site and in-situ sensing of pesticide residue and the lack of effective technical methods,we have established the novel electrochemical bio-nano sensing methods for OPs based on bio-specific identification and nano-efficient sensitization mode,studied the flexible and wearable electrochemical bio-nano sensing devices with self-adaptive ability for irregular crop surfaces,and investigated the feasibility for OP residue on crop surfaces.Main research contents and results are as the following:(1)Investigation of sensing mechanisms of OP molecules based on the electrochemical properties of bio-nano interfacesAiming at the difficulty of low content of pesticides and high amount of interferents on crop surfaces,the highly specific and sensitive bio-nano interfaces were constructed and the sensing mechanisms for OPs based on the electrochemical properties were revealed.We firstly established the bio-nano interface of acetylcholinesterase(ACh E)-metallic molybdenum sulfide(Mo S₂),and explored the electrochemical sensing mechanism for paraoxon at this interface based on enzyme inhibition.The activity of ACh E could be inhibited by paraoxon,consequently leading to the reduced output of reactive intermediates.Mo S₂ can accelecrate the electron output from the intermediate oxidation and offer the electronic channel.Therefore,paraoxon can be detected by comparing the current at Mo S₂ nano-interface before/after the inhibition.We also established the bio-nano interface of organophosphorus hydrolase(OPH)-three-dimensional porous graphene,and explored the electrochemical sensing mechanism for methyl parathion at this interface based on enzyme hydrolyzation.OPH can shear the P-O bonds of methyl parathion,producing the electroactive p-nitrophenol,consequently causing the electron transfer on graphene nano-interface due to the the electronic channel of graphene.Therefore,methyl parathion can be directly detected by comparing the current at graphene nano-interface.(2)Fabrication and performance study of integrated electrochemical sensing devices for OP moleculesIn order to overcome the poor operability and the in-situ analysis impossibility of traditional electrochemical discrete three-electrode detection system,the integrated electrochemical sensing devices were designed based on screen-printed technique,which could make the on-site and rapid sensing of OPs possible.To further improve the sensitivity,two-dimentional titanium carbide nano-interface was firstly constructed on working electrode surface,and then was used as the self-reducing template for the growth of gold-palladium bimetallic nanoparticles,consequently establishing the multi-dimensional nanocomposite interface of titanium carbide/gold-palladium bimetallic nanoparticles.The obtained bimetallic nanoparticles could be spontaneously grown in 5 min,which were easy to prepare and shape-controlled.They also showed excellent synergistic catalytic ability with enzyme,which could further improve the sensing performance.Using paraoxon as the model pesticide,this method showed good anti-interference property with the detection limit of 1.75 ng/L.Pear and cucumber samples were used to evaluate the feasibility of this method.The recoveries of paraoxon are from87.93%to 111.02%with the relative standard deviation from 1.08%to 6.37%(n=3),which could offer a reliable technical method for on-site sensing of paraoxon.(3)Optimization and evaluation of semisolid electrolytes for in-situ organophosphorus pesticide analysis at solid-phase interfacesIn order to solve the problem of the mass transfer of pesticide molecules on solid surfaces from target surface to sensing interface during the in-situ analysis,the biocompatible semisolid electrolytes were designed and developed.The semisolid electrolytes were fabricated using gelatin/agarose as the gelling agent,kalium salt and sodium salt as the electrolytes,respectively.We also explored their gel strength,diffusion ability,the influence for enzyme activity,and electrochemical performance.Results showed that the optimized semisolid electrolyte was formed with 2.5 wt%gelatin as the gelling agent,100 m M potassium chloride and 100 m M potassium phosphate monobasic as the electrolyte.After that,the primary in-situ analytical method for methyl parathion on solid surfaces was established using the integrated screen-printed three-electrode device as the sensing device,modified with OPH and the prepared semisolid electrolyte at p H 9 and with the diffusion time of 8 min.This method could be applied for the on-site and in-situ sensing of methyl parathion on the polluted glass,plastic,wooden table and aluminum foil surfaces.(4)Flexible and wearable biosensor for the in-situ OP analysis on crop surfacesAiming at the problem that sensing devices can not fit the irregular crop surfaces effectively,a flexible and wearable biosensor for the on-site and in-situ sensing of pesticide residue on crop surfaces was established.The integrated serpentine three-electrode system was prepared via the laser-induced graphene(LIG)technology.After transferred by polydimethylsiloxane,the flexible and stretchable LIG-based electrode was obtained,which can be well adapt to the irregular surface of crops,such as leaves and fruits.To decrease the working potential and increase the sensitivity,the bio-nano interface of OPH-LIG/Au nanoparticles was established.After equipped with gelatin semisolid electrolyte and hand-held electrochemical workstation,our flexible and wearable biosensor can effectively recognize the methyl parathion,with the detection limit of 0.26 ng/cm².The pesticide residue data can be transmitted to a smartphone device wirelessly in real time via Bluetooth in hand-held electrochemical workstation,which enable the on-site and in-situ electrochemical analysis of pesticide on the surface of spinach leaves and apple fruits.