Magnetic Separation-based Nanobiosensors For Detection Of Foodborne Pathogen And Antibiotic Residues In Poultry

Along with the substantial growth of poultry production,the safety of poultry products has gained ever increasing attention in past two decades.Among various contaminants,foodborne pathogens and antibiotic residues are major risk factors in poultry products.Therefore,it is of great importance to detect foodborne pathogens and antibiotic residues with high rapidity,specificity and sensitivity.Biosensors are now recognized as one of the most promising detection tools to meet these needs.However,the biosensors’ability to deal with complex food samples should be further improved.Magnetic separation can help isolate targeted analytes from food matrices to improve the performance of biosensors.Meanwhile,the cooperation of nanomaterials into signal transducing can avoid the complicated signal generation/amplification and improve the stability of the biosensor system.Therefore,this dissertation focuses on the development of different types of nanomaterial-based biosensors（e.g.fluorescent,magnetic relaxation switching（MRS）and colorimetric biosensors）that integrate magnetic separation with nanomaterials labeling for the detection of Salmonella and enrofloxacin（ENR）residues in poultry.The ability of signal generation of magnetic nanoparticles（MNPs）was also explored.Furthermore,the developed fluorescent nanobiosensor was also coupled with a portable detector to improve its in-field application.The main research contents are as follows:（1）A fluorescent biosensor based on magnetic separation,quantum dots（QDs）labeling and CRISPR-Cas12a cleavage was developed for Salmonella detection.Firstly,streptavidin（SA）and single-stranded DNA（ss DNA）S₂ were modified on the surface of MNPs and QDs to prepare MNPs-SA and QDs-S₂ probes,respectively.MNPs-QDs complexes could be formed by using a complementary ss DNA modified with biotin（biotin-S₁）as the linkers.In the presence of target DNA,the binding of QDs-S₂ to MNPs-SA was inhibited due to the non-specific cleavage of biotin-S₁ caused by the activated CRISPR-Cas12a system,resulting in a decreased fluorescent signal.Benefitting from magnetic separation,the fluorescence properties of QDs and the high specificity and cleavage activity of the CRISPR-Cas12a system,the developed biosensor was sensitive to Salmonella with limits of detection（LODs）of 1 n M and1.3×10² CFU m L^-1 for Salmonella DNA and Salmonella cells,respectively.By integrating the advantages of MNPs,QDs and the CRISPR-Cas12a system,the fluorescent biosensor provides a new idea for foodborne pathogen detection.（2）To avoid the drawbacks of repeated separation and washing,as well as the requirement of strict preservation of QDs labels in the fluorescent biosensor in part（1）,a CRISPR-Cas12a-based MRS biosensor using large MNPs for magnetic separation and small MNPs for signal generation was proposed for Salmonella detection.The binding of the two kinds of MNPs using biotin-S₁ as the linkers was inhibited through the target-activated CRISPR-Cas12a cleavage system as presented in part（1）.After separating the MNPs of large size from those of small size,the transverse relaxation time（T₂）of the water molecules around the small MNPs was measured for Salmonella detection.Benefitting from the high specificity of the CRISPR-Cas12a system and the high sensitivity of MRS detection,this biosensor allowed for sensitive detection of Salmonella with LODs of 1.3×10² CFU m L^-1 and 1.8×10³ CFU m L^-1 in pure culture and chicken meat extracts,respectively,without multiple washing and magnetic separation.（3）A fluorescent biosensor based on MNPs and QDs was developed for ENR detection.Two sensing elements,antibody functionalized MNPs（immuno-MNPs）and ENR-bovine serum albumin conjugates modified QDs（QDs-BSA-ENR）were designed and fabricated.The fluorescent biosensor was designed based on the competitive binding of QDs-BSA-ENR and target ENR towards immuno-MNPs.It allowed for sensitive detection of ENR with a LOD of 0.94 ng m L^-1.For chicken meat tests,a 5-sulfosalicylic acid-based pretreatment method was adopted to extract ENR from chicken meat samples and reduce non-specific adsorption caused by complex food matrices.Benefitting from MNPs for target capture and separation,the biosensor displayed satisfactory performance in chicken meat with a LOD of 14.1μg kg^-1 and recovery rates ranging from 83.3%-101.6%.The whole analytical procedure from food sampling to result report could be finished in less than 1.5 h.This nanobiosensor showed high potential for rapid and low-cost detection of ENR residues in poultry to enhance food safety.（4）To simplify the detection procedure,shorten the analysis time and also take the full advantages of MNPs,e.g.sample separation and signal generation,a label-free biosensor based on target-induced immuno-MNPs aggregation was proposed for rapid and facile detection of ENR.Firstly,the underlying mechanism of ENR-induced immuno-MNPs aggregation was explored which might be a result of the increased surface hydrophobicity.Then,based on the differences on magnetic separation efficacy and size,turbidity and gray-scale measurements were performed for ENR detection.The biosensing methods exhibited satisfactory detection performance with LODs of0.79 ng m L^-1 and 7.77 ng m L^-1 for turbidity and gray-scale measurements,respectively.Besides,the detection could be finished in 15 min with high simplicity,showing its potential for in-field application.（5）To meet the requirement of in-field application,the developed fluorescent nanobiosensor was coupled with a portable detector for ENR detection in poultry production chain.It exhibited satisfactory detection performance in poultry samples with the LODs of 1.5 ng m L^-1,10μg kg^-1 and 3.73μg kg^-1 for ENR in drinking water,feeds and chicken breasts,respectively.Compared to the standard HPLC-MS/MS and ELISA methods,the biosensor had an accuracy of approximately 83%for 66 chicken breast samples,showing its great potential for ENR detection in poultry.