The Construction And Application Of Novel Sensing Interfaces For Pesticide Determination And Bioactivity Research

Use of pesticides contributes positively to agricultural development.However,excessive use is associated with serious risks to environment and human health.Hence,scientific researchers are facing by these two challenges:Firstly,development of rapid,effective and credible pesticide-residual analytical methods to monitor food quality process.Secondly,to discover high potent pesticides of low toxicity and residue to control the environmental and health risks.Combining high selectivity and molecular recognization ability of interaction between target protein and its substrate with sensitivity and convenience of electrochemical detection techniques,biosensor is considered to be hot topics of current research in pesticide residue detection.It is significant to develope in-vitro sensitive analyical methods for pesticides screening and discovery.For both pesticide-residual detection and drug screening,a construction of interface for immobilization of biomolecules is a key factor to influence sensitivity and stability of sensing signal.Several bionic interfaces were constructed in this paper,using acetylcholinesterase(ACHE) and D1 protease(CtpA) as target proteins.Focusing on development of new methods for pesticide residue detection and drug sensitivity comparison based on target protein molecular recognization by the technology of surface plasmon resonance(SPR) and electrochemistry,we carried out some researches as follow.1.Molecular recognization of acetylthiocholine to immobilized AChE on multiwall carbon nanotube-cross linker chitosan compositeChitosan provided a satisfied microenvironment to retain the biological activity of AChE with glutaraldehyde as cross-linker.Because of the introduction of multiwall carbon nanotube,the oxidation potential of thiocholine was much reduced.The immobilized AChE had greater affinity for acetylthiocholine(ATCI) and excellent catalytic effect in the hydrolysis of ATCI,with a K_m^app value of 132μM.Under optimum conditions the amperometric current increased linearly with increasing concentration of ATC1 in the range 2.0-400.0μM.This method showed very good reproducibility, sensitivity and acceptable stability.It is a promising new tool for characterization of enzyme inhibitors and for pesticide analysis.2.An composite interface based on immobilization of acetylcholinesterase on a multiwall carbon nanotube-cross-linked chitosan for pesticide detection and sensitivity comparisonPresence of pesticides in solution will reduce the interaction between ATCI and immobilized AChE on a multiwall carbon nanotube-cross-linked chitosan composite,which resulted in an electrochemical signal reduction caused by thiocholine.Based on competition strategy,the inhibition of triazophos was proportional to its concentration in two ranges,from 0.03 to 7.8μM and 7.8 to 32 μM.Pesticides of carbaryl,malathion and dimethoate were selected to study their inhibition efficiencies to ACHE,which is illuminated the establishment of a speedy comparison for pesticide sensitivity by electrochemical technique.The constructed biosensor processing prominent characteristics and performance such as simple fabrication,fast response,acceptable stability and accuracy has potential application in the characterization of enzyme inhibitors and detection of toxic compounds to enzyme.3.Composite assembly of silver nanoparticles with biotinylated AChE for pesticidal sensingUsing avidin as a linker,a biosensor has been devised by immobilization of biotinylated AChE on a biotin-terminated and 11-mercapto-l-undecanolmixed self-ssembly monolayer.silver nanoparticles was introduced by the electrostatic interaction between silver nanoparticles and avidin.Under the optimum conditions a quantitative measurement of organophosphate pesticide dimethoate was achieved with the linear range of 0.05μM to10.0μM and the correlation coefficients of 0.9983.The detection limit was 0.01μM,which corresponds to a 10%decrease in signal.4.Interaction research between gold nanoparticles labeled carbamate and AChE by SPRTwo carbamate inhibitors with different ether linkages and the terminal lipoate were synthesized and labeled with gold nanoparticles.With the signal amplification of AuNPs,the specific interactions between the AuNPs labeled carbamate inhibitors and the immobilized ACHE on sensor chip surface were readily examined by SPR.Using 1:1 fitting model,the association/dissociation rate constants were first obtained for the binding interaction between carbamate inhibitors and ACHE.This AuNPs labeling strategy is versatile and may be applicable for the competitive SPR kinetic assay of the interaction between small molecule inhibitors and their target proteins with a high sensitivity.5.Signal enhancement by AChE stimulated in situ growth of gold nanoparticles for SPR technology-based sensing of inhibitor.The hydrolysis of acetylthiocholine chloride(ATC1) catalyzed by AChE can yeild a reducing agent thiocholine that stimulates the formation of AuNPs in the presence of HAuCI4,which caused a SPR signal.The formation of the AuNPs was inhibited by triazophos,thus enabling a sensitive determination for AChE inhibitor by SPR technology.The inhibition of methomyl on AChE was proportional to its concentration in the range of 0.5-14.0μM,with detection limit of 0.05μM. Inhibitor determination was achieved without labelling or modification for target protein ACHE,which is appropriate for enzyme-based detection.Such a simple and convenient strategy may find wide potential applications in biosensors,biocatalysis and durg screen.6.Interaction research between D1 protease and ferrocene labeled 24 peptide and application in inhibitor sensitivity comparison by electrochemical technique. A novel interface embedded in situ gold nanoparticles in chitosan hydrogel was constructed for CtpA immobilization by one-step electrochemical deposition.Ferrocene labeled 24 peptide was acted as electrochemical probe because of its redox activity.The current response that arose by ferrocen displayed interaction between immobilized CtpA and 24 peptide.The current response was proportional to concentration of ferrocene labeled 24 peptide in the range of 2.0-65.0μM.Inhibitor sensitivity comparison was achieved by comparing inhibition rate of three kinds of CtpA inhibitors at the same concentration.7.Interaction research between D1 protease and gold nanopartivcle labeled 24 peptide by SPR: Kinetic analysisCtpA was immobilized on a carboxyl methyl dextran interface by acylamide bond to study the interaction between CtpA and gold nanoparticle labeled 24 peptide by SPR.The kinetic data was obtained with K_a= 3.08×10²(Ms)^-1,K_d=6.24×10^-3 s^-1.Presence of inhibitor in solution will reduce the interaction between CtpA and 24 peptide on a multiwall carbon nanotube-cross-linked chitosan. composite,resulting in SPR signal reduction.Based on competition fitting model,the kinetic data including association rate constant,dissociation rate constant were obtained,which provided a new method for inhibitor sensitivity comparison and screening.