Novel Photoelectrochemical And Temperature Biosensors Based On Nucleic Acid Signal Amplification For Detection Of Organophosphorus Pesticides

Organophosphorus pesticides have strong neurotoxicity.And it can inhibit the activity of acetylcholinesterase in the nervous system resulting in serious human and animal health problems even at low concentrations.Biosensor is a microanalysis method that uses biologically active materials（enzymes,proteins,DNA,antigens,antibodies,etc.）to organically combine with physical and chemical transducers.Due to its simplicity,speed,low cost,high sensitivity and good selectivity,many biosensors was used to detect organophosphorus pesticide residues.New nucleic acid signal amplification technologies（such as rolling circle amplification,hybridization chain reaction,chain substitution amplification reaction,etc.）play a vital role in trace detection,which has accelerated the further development of biosensors.The DNA hydrogel is a good connection between DNA nanotechnology and biotechnology,providing broad prospects for its application.This paper mainly studies the design of nucleic acid signal amplification technology and the control of biomolecules by DNA hydrogel,and constructs three new types of biosensors to realize the sensitive detection of organophosphorus pesticides.The specific work is carried out as follows:1.This work devised a photoelectrochemical（PEC）aptasensing platform for sensitive detection of organophosphorus pesticide（malathion used in this work）based on dissolution of core-shell MnO₂ nanoflower@CdS（MnO₂NF@CdS）by the as-produced thiocholine（TCh）from the butyrylcholinesterase-acetylthiocholine system,accompanying with the target-triggered rolling circle amplification（RCA）.The core-shell MnO₂NF@CdS with excellent PEC performance was synthesized and employed as a photo-sensing platform.The corresponding aptamer binds to the capture DNA（cDNA,with primer fragment）modified on the magnetic beads.Upon malathion introduction,aptamer was detached from the magnetic beads,while cDNA remained on the beads.The primer fragment in cDNA can trigger RCA reaction to form a long single-stranded DNA（ss DNA）strands.Furthermore,a large number of BChE were assembled on the long ss DNA strands through the hybridization with the S₂-Au-BChE probe.Thereafter,TCh generated from hydrolysis of ATCh by BChE can reduce MnO₂ NF（core）to Mn²⁺and release the CdS nanoparticles（shell）from the platform electrode,subsequently significantly enhance the PEC signal.Under optimal conditions,the proposed aptasensor exhibited high sensitivity for malathion with a low detection limit of 0.68 pg mL^-1.Meanwhile,it also presents outstanding specificity,reproducibility,and stability.2.A highly sensitive photoelectrochemical（PEC）sensing platform for malathion detection based on the biocatalysis-induced formation of BiOBr/Bi₂S₃ semiconductor heterostructures.Initially,a double-stranded DNA consisting of an initiator strand and the malathion aptamer was immobilized on magnetic beads surface.The target malathion could competitively bind with its aptamer and liberate the initiator strand from the double-stranded DNA.Subsequently,the initiator strand triggered a hybridization chain reaction（HCR）to produce a long DNA concatemer containing the sequence of G-quadruplex structure.The DNA concatemer was then endowed with horseradish peroxidase（HRP）-like activity via the formation of hemin/G-quadruplex complexes and binding with MnTMPy P.The HCR-synthesized HRP-mimicking DNA concatemer was employed to catalyze Na₂S₂O₃ conversion to H₂S with the aid of H₂O₂.The generated H₂S could react with BiOBr nanoflowers（BiOBr NFs）modified on an indium tin oxide electrode,resulting in the in situ formation of BiOBr/Bi₂S₃ heterostructures with enhanced photoelectric conversion efficiency.The BiOBr/Bi₂S₃ heterostructures could generate a strong photocurrent signal to indirectly indicate malathion concentration.Under the optimal conditions,a wide linear range between the malathion concentration and photocurrent intensity(0.001-1000 ng mL^-1)and a limit of detection as low as 0.12 pg mL^-1 were achieved.3.A simple thermal aptasensing platform was devised for sensitive detection of organophosphate pesticides（malathion as a model target）based on the efficient self-heating reaction of warm pad with a switchable target responsive enzyme-encapsulated three-dimensional（3D）DNA hydrogel by using a portable thermometer as signal readout in this work.A target-responsive DNA hydrogel by using aptamer as a cross-linker was synthesized with catalase（CAT）trapped inside.In the absence of malathion,CAT could not react with the H₂O₂ due to the barrier effect of DNA hydrogel.In contrast,the existence of target malathion would open the 3D network and lots of CAT was released from the hydrogel,which could efficiently convert the H₂O₂ to O₂ molecule.The product O₂ is the critical conditions for the self-heating of warm pad.Thereafter,the temperature enhanced with the increasing amount of O₂.Under optimal conditions,the as-proposed thermal biosensor exhibited high sensitivity for malathion with a linear range from 0.1 to 1000 ng ml^-1 and a low detection limit of 0.03 ng ml^-1.Overall,such a sensing platform with outstanding specificity,reproducibility,and stability provides a new perspective for detection of organophosphate pesticides.