Construction Of Electrochemiluminescence Biosensor Based On Organic Framework Material And CRISPR/Cas12a Signal Accumulation Strategy

In agricultural production,the use of pesticides contributes to improve the yield and quality of crops.However,excessive pesticide residues may be causing serious threat people’s life and health,so the detection of pesticide residues plays a crucial role in ensuring food safety.Electrochemiluminescence（ECL）is the phenomenon of chemiluminescence induced by electrochemical reactions that occurred on or near the electrode surface.ECL analysis technology combined the sensitivity of luminescence analysis with the controllability of electrochemical analysis.The traditional ECL pesticide residue biosensor was difficult to meet the requirements of sensitive detection of trace target due to lack of efficient amplification strategy for target molecules.Aptamer can use as a medium for combining pesticide residue biosensors with nucleic acid amplification technology,thereby achieving cyclic amplification of target.The operating principle of these existing ECL aptasensors for pesticide residue detection was mainly based on the ECL response that induced by the specific interaction between the target and the aptamer modified on the sensing interface.Nevertheless,there are still many shortcomings:（1）The interaction between the target and the aptamer occurred on the heterogeneous phase interface between the electrode and the solution,so the recognition ability of the aptamer was inefficient due to the steric hindrance of the electrode surface.（2）This detection method only utilized the effect of interaction between pesticide small molecules and aptamers toward ECL material,and lack of cyclic amplification strategy of the target,resulting in relatively low detection sensitivity of pesticide residues.Nanomaterials play a positive role in improving sensitivity of ECL biosensor.So,we combined the following two ways:（1）preparation of nanomaterials with excellent ECL performance to construct efficient ECL sensing platform.（2）Design of the magnetic spherical nucleic acid that can efficiently recognize pesticide residues to improve the recognition and conversion efficiency of the target.Finally,the target conversion strategy combined with CRISPR/Cas12a mediated signal amplification strategy to construct efficient ECL biosensor for the detection of pesticide residues in this paper.The research work of this paper mainly consists of the following parts:1.ECL covalent organic framework coupling with CRISPR/Cas12a-mediated biosensor for pesticide residue detection.The trace detection of pesticide residue becomes particularly important since increasing attentions have been attached to food safety.Herein,we developed an ECL covalent organic framework（COF）based-biosensor for trace pesticide detection coupling with CRISPR/Cas12a-mediated signal accumulation strategy.Specially,a target conversion was carried out with an aptamer-assembled magnetic spherical nucleic acid,which can convert acetamiprid to activator DNA,activating the CRISPR/Cas12a to make Fc-DNA far away from electrode for ECL response.The perylene-3,4,9,10-tetracarboxylic COF（PTCA-COF）with stable and strong ECL was synthesized by a condensation reaction between the perylene-3,4,9,10-tetracarboxylic dianhydride（PTCDA）and melamine（MA）,due to the highly ordered arrangement of the PTCDA luminescence units among COF structure and the pore confinement effect.Using this established biosensor,the acetamiprid could be determined sensitively with a wide linear range from 100 pmol/L to 100μmol/L and a limit of detection of 2.7 pmol/L.Moreover,this work expands the application of CRISPR/Cas 12a and provides an avenue for target amplification of pesticide residue molecule.2.ECL biosensor based on triple-helix combined with cascade-signal amplification strategy mediated by self-cutting DNAzyme and CRISPR/Cas12a for detection of pesticide residue.In this work,we combined the design of a triple-helix molecular switch with the cascade signal amplification strategy mediated by target induced self-cutting DNAzyme and CRISPR/Cas12a to improve the conversion efficiency of the target to construct ECL biosensor for the detection of acetamiprid.Specifically,in the presence of the target,the triple-helix molecular switch was opened,exposing the primer and further initiate rolling circle amplification reaction to generate numerous DNAzymes with self-cutting activity.Subsequently,a large number of activator DNA was output that assisted by coenzyme factor Zn²⁺,achieving preliminary amplification of the target.The activator DNA further activated the side chain shearing activity of CRISPR/Cas12a to efficiently cut the single-stranded DNA signal probe modified on the electrode interface,realizing the signal output and cascade amplification of target.At the same time,we used the metal-organic frame material ZIF-8 with ordered frame structure as the limiting space of the ECL molecule perylene（Pe）,which（ZIF-8@Pe）improved the disordered stacking state of luminescent molecules and weaken the aggregation-induced quenching effect（ACQ）,achieving the eccellent ECL performance of ZIF-8@Pe.The established biosensor displayed a wide linear range from 1 pmol/L to 100μmol/L and a limit of detection of 335 fmol/L for acetamiprid.