Construction And Application Of Highly Sensitive Biosensing Interface For Pesticide Residues

In this thesis,a series of functional nanomaterials,such as polymers,noble metal nanomaterials,semiconductor nanomaterials,carbon-based nanomaterials,metal-organic framework materials,bimetallic magnetic nanoparticles and their composite materials,have been prepared to construct a variety of electrochemical biosensor interface.The effects of size and morphology of functional materials on sensing performance were investigated.The selective recognition of the target and catalytic performance by the interface composition and properties were discussed.A variety of electrochemical biosensors were constructed using sensing strategies such as dual amplification,energy resonance transfer,dual inhibition,dual mode and co-reaction enhancers.By optimizing the measurement conditions,a variety of pesticide residue analysis methods were established and practical application research was carried out.The main contents include:1.Based on layer-by-layer template technology and catalytic amplification,an electrochemical aptasensor was proposed for acetamiprid detection.Herein,the signal probe of the ferrocene（Fc）-based hollow polymeric nanospheres（Fc-HPNs）were prepared with electrostatic adsorption between anionic poly（acrylic acid）and hyperbranched cationic polyethylenimine.Remarkably,the Fc-HPNs played dual roles:as nanocarriers,to significantly increase the load amount of Fc;and as nanoreducers,to effectively catalytic reduction by ascorbic acid for further signal amplification.Surprisingly,this proposed aptasensor exhibited excellent sensitivity with a linear range from 1.0×10^-81.0×10^-1515 mol/L and limit of detection down to 3.3×10^-16 mol/L,which opened a novel avenue for monitoring the acetamiprid.2.Based on energy resonance transfer between CdS/MoS₂ nanospheres and Ag/CQDs,an electrochemiluminescence aptasensor was proposed for chlorpyrifos detection.MoS₂/CdS nanospheres synthesized by biomolecular assisted method have excellent biocompatibility and electrochemiluminescence properties.Ag/CQDs,as receptors for energy resonance transfer,can improve the sensitivity of detection.This proposed aptasensor exhibites excellent sensitivity with a linear range from 1×10^-151×10^-8 mol/L and limit of detection down to3×10^-16 mol/L.The aptasensor has the characteristics of high specificity and high sensitivity,which provides a new idea for the detection of chlorpyrifos.3.Based on the principle of double inhibition,an electrochemiluminescence sensor was constructed for the highly sensitive detection of glyphosate.First,a graphene-gold nanoparticle composites（rGO-Au）was synthesized to increase the specific surface area of the electrode as an enzyme capture substrate.Secondly,the luminol-gold nanoparticle-L-cysteine-Cu²⁺composite(Lu-Au-Lcys-Cu²⁺)was synthesized as a luminescent reagent and a peroxidase to improve the detection signal.Hydrogen peroxide（H₂O₂）was obtained as a co-reactant of Lu-Au-Lcys-Cu²⁺by two-step enzymatic hydrolysis using acetylcholine chloride as a substrate.The hydrolysis reaction in the first step is that acetylcholinesterase catalyzes the chlorination of acetylcholine to give acetic acid and thiocholine.The second step of the hydrolysis reaction is the production of dithiobischoline and H₂O₂ by choline oxidase catalyzed by choline oxidase in the presence of dissolved oxygen.When glyphosate is applied to the surface of the above modified electrode,glyphosate can inhibit the activity of acetylcholinesterase;at the same time,it can form a complex with Cu²⁺to make it detach from the surface of the electrode and lose its catalytic effect.The detection range of the sensor we constructed for glyphosate was 0.001¹.0mmol/L,and the detection limit was 0.5 nmol/L.The sensor based on Lu-Au-Lcys-Cu²⁺and dual inhibition strategy achieves highly sensitive detection of glyphosate.4.Based on NH₂-MIL-88（Fe）as the nanocarrier and signal enhancer,a novel electrochemiluminescence aptasensor was constructed for the detection of malathion.At first,based on the specific surface area,good conductivity and biocompatibility of Au NPs@rGO,a large number of binding sites are provided for the modification of aptamers.Then,Fe（Ⅱ）from NH₂-MIL-88（Fe）as the signal enhancer,could react with S₂O₈^2-to generate Fe（Ⅲ）and SO₄^·-.And the Fe（Ⅲ）can be reduced under the electrochemical reaction.Thus,the cycle-type reaction was come true to produce amounts of SO₄^·-,which could react with CdTe QDs to realize a relative stronger ECL signal.The detection range of the sensor we constructed for malathion was 1.0×10^-61.0μg/L,and the detection limit was 0.3 pg/L.Importantly,NH₂-MIL-88（Fe）as the nanocarrier and signal enhancer has provided a direction for ultrasensitive target analysis.5.Based on electrochemiluminescence and colorimetric dual mode,a biosensor was constructed for the detection of malathion.The gold nanoparticles are assembled into a three-dimensional gold hydrogel by dopamine induction,which has excellent biocompatibility,electrical conductivity,large specific surface area,and porosity.The combination of a three-dimensional gold hydrogel and luminol to form a composite material has excellent ECL properties.The malathion aptamer and alkaline phosphatase were supported by CuFe₂O₄magnetic nanocrystal clusters（CuFe₂O₄ MNCs）with peroxidase-like activity.CuFe₂O₄MNCs could catalyze the hydrolysis of alkaline phosphatase.The ascorbic acid produced by the enzymatic hydrolysis has a dual function:（1）as an inhibitor of hydrogen peroxide in the luminol co-reactant in the electrochemiluminescence sensor;（2）as a catalyst for methylene blue in the colorimetric mode.This proposed aptasensor exhibited excellent sensitivity with a linear range from:electrochemiluminescence:1.0×10^-91.0×10^-15 mol/L(detection limit5.0×10^-16 mol/L);UV absorption spectrum:1.0×10^-61.0×10^-12 mol/L(detection limit7.0×10^-13 mol/L).Benefiting from the different mechanisms and relatively independent signal transduction in the dual mode described above,our dual-mode sensor improves the accuracy and sensitivity of the detection.6.Based on CoFe₂O₄ magnetic nanoparticles（CoFe₂O₄ MNPs）as a co-reaction enhancer,an electrochemiluminescence sensor was constructed for the sensitive detection of chlorpyrifos.At first,we prepared CdS@Au NPs@rGO composite material,which can load a large amount of CdS nanocrystals based on Au NPs@rGO.Secondly,we prepared CoFe₂O₄MNPs with extremely large specific surface area and magnetic properties.Here,CoFe₂O₄MNPs can play a dual role:as a carrier,loading acetylcholinesterase;as a enzymatic reactant,promoting acetylcholinesterase catalyzes the production of thiocholine by acetylcholine chloride.Further,the thiocholine formed by the enzymatic reaction can increase the electrochemiluminescence intensity as a co-reaction enhancer of the CdS@Au NPs@rGO-S₂O₈^2-system.The detection range of the sensor we constructed for chlorpyrifos was 1.0×10^-61.0×10^-12 mg/L,and the detection limit was 6.0×10^-1313 mg/L.Thiocholine as a co-reaction enhancer of the CdS@Au NPs@rGO-S₂O₈^2-system also extends the application of acetylcholinesterase.