Highly Reliable Methods For The Detection Of Typical Pesticide Residues In Apple Based On Surface-enhanced Raman Spectroscopy

Apples are one of the most popular staple fruits.Pesticide residues in apples have great influence to consumer health.In order to ensure food safety,it is of great practical significance to detect pesticide residues in apples and determine whether they meet relevant standards.Surface-enhanced Raman spectroscopy（SERS）has the characteristics of high sensitivity,simple operation,and excellent spectral fingerprint recognition ability.It has been widely used in molecular detection applications and is an effective way to realize pesticide molecular detection.This research focuses on solving the problem that the single noble metal gold or silver in the existing SERS substrate cannot satisfy the strong Raman enhancement effect and high chemical stability at the same time,and the bimetallic nanomaterial with high enhancement factor and repeatability was produced as the SERS substrate for the detection of thiram.The Raman spectra of non-target molecules in complex detection systems are prone to overlap and interfere with the Raman spectra of target molecules.Therefore,SERS aptamer sensors and immunosensors mediated by nitrile organic molecules were designed to achieve anti-spectral interference detection of acetamiprid and imidacloprid,respectively.Organic molecules as Raman tags have the disadvantages of high toxicity and complex follow-up operations.Hence,a non-toxic SERS immunosensor mediated by inorganic molecules and facile operation process has been developed to detect carbendazim.The main research contents are as follows:（1）The preparation of SERS substrate with both high stability and strong SERS acitivity for the detection of thiram in apple.To solve the shortcoming that a single noble metal gold could not satisfy the strong Raman enhancement effect or silver may not meet the requirement for high chemical stability,this study proposed a solution of preparing bimetallic nanomaterials as a SERS substrate.High-purity three-dimensional gold nanodendrites（Au ND）without organic contamination were prepared by a simple,efficient,and rapid electrodeposition method,and then silver nanoparticles（Ag NP）were modified on the surface of Au ND to make Ag-Au NDs substrate.Thiram pesticide molecule has a disulfide bond group,therefore it can be directly bonded to the surface of the SERS substrate through chemical bonds to achieve direct detection of thiram.The results showed that the SERS substrate had good Raman spectrum enhancement performance.The obtained thiram SERS spectrum was used to establish the standard curve.The detection range was 0.12-24 mg/L,and the limit of detection value was 86.1μg/L.At the same time,the standard addition method was used for the establishment and detection of the thiram standard curve in apple samples.The recovery rates for 0.5and 1 mg/L thiram were 96.7%and 94.8%,respectively.This study indicated that the constructed label-free direct detection method can not only synthesize the SERS substrate simply and efficiently,but also realize the rapid detection of thiram in apple samples.（2）The construction of SERS aptamer sensor system mediated by the specific Raman signal in cell’s Raman-silent region for the anti-interference detection of acetamiprid in apple.To solve the problem that the commonly used Raman tags in sensors are easily interfered by the Raman spectra of other non-target molecules in complex detection systems,this study proposes utilizing nitrile organic molecules with Raman characteristic peaks located in the biological Raman-silent region as Raman tag.Then,gold nanoparticles（Au NPs）connected with nucleic acid aptamers and nitrile organic Raman tag molecule were used as signal probes.Silver nanoparticles which were fabricated on silicon wafers forming Ag NPs@Si substrates and connected with complementary DNA,were applied as a competitor of acetamiprid molecule.When acetamiprid was present,acetamiprid would bind specifically to the aptamer,causing the SERS spectra intensity of signal probe attached to the Ag NPs@Si substrate weaken.The obtained Raman spectrum intensity decreased accordingly,and the detection of acetamiprid concentration could be realized.The results showed that the detection range of the established SERS aptamer sensor is 25-250 n M,and the limit of detection value is 6.8 n M.Anti-interference and reproducibility tests indicated that this sensor has good detection performance.At the same time,the acetamiprid content in apple juice samples was detected by the standard addition method,and the recovery rate was86.1%-100.3%.This study shows that it is feasible to design a sensor based on nitrile organic molecules as Raman tag to detect acetamiprid in apple samples.（3）The fabrication of a SERS immunosensor system mediated by nitrile organic molecules and antibody with specific capture ability for the anti-interference detection of imidacloprid in apple juice.Gold nanorod-coated silver nanocubes（Au NR@Ag）with uniform particle size were connected with nitrile organic Raman tags and imidacloprid antigens.And Fe₃O₄ magnetic nanoparticles was modified with imidacloprid antibodies.When imidacloprid was present,the imidacloprid molecule competitively binded to the antibody,so that the binding amount of antigen and antibody was depended on the concentrations of imidacloprid.The SERS signal intensity of Raman tag on the surface of the Fe₃O₄ nanoparticles after magnetic separation would decrease with the increase of imidaclorpid concentration.The results showed that the detection range of imidacloprid was 10-400 n M,and the limit of detection value was 9.58 n M.At the same time,the spectral anti-interference ability test of this SERS immunosensor indicated that even in complex organic molecules,nitrile Raman tags can also obtain excellent spectral anti-interference ability.In actual apple samples,the recovery rate of imidacloprid by standard addition method was 96.8%-98.6%.The results showed that the competitive SERS immunosensor had good spectral anti-interference performance in complex systems,and can be used for trace detection of imidacloprid residues in apple juice samples.（4）The construction of a SERS immunosensor system mediated by green and facile synthesized nitrile inorganic nanomaterials for the anti-interference detection of carbendazim in apple.To overcome the shortcomings of organic molecules as Raman tags in sensor,such as high toxicity and complex follow-up sensor preparation steps,this study proposes the use of non-toxic Prussian blue inorganic molecules as Raman tag to simplify the fabrication process and eliminate the possibility of error.The Prussian blue metal frame structure was modified on the surface of the gold nanoflower,and the positively charged polylysine was adsorbed by electrostatic attraction,followed by the adhesion of negatively charged antigen.Then,the antibody was modified on the surface of Fe₃O₄ by the amide reaction.The competitive binding effect of antigen and carbendazim to antibody was used to establish a highly sensitive detection system.The results showed that the detection range of carbendazim was 0.1-100 n M,and the limit of detection value was 0.02 n M.At the same time,the immunosensor was applied to detect carbendazim content in apple samples,and the recovery rate ranged from 91.8%to 112.8%,indicating that the SERS immunosensor can realize highly sensitive detection of carbendazim content in apple samples.