Construction Of Label-free Aptasensor And The Study Of Their Highly Sensitive Detection For Multiclass Contaminants In Complex Matrices

With the improvement of people's quality of life,the safety of food and the environment has become more and more concerned by consumers,especially the residues of various pollutants in food and the environment.At present,more and more toxic and harmful substances reduce the nutritional value and quality of food or cause different degrees of harm to the human body.However,the matrix of food and environmental samples is very complicated,and the content of various types of pollutants is extremely low.Multiple types of pollutants often exist in the matrix of a sample.Therefore,the detection process is easily interfered by the matrix and false negative results appear,which makes the detection process Facing huge challenges.Therefore,it is necessary to adopt effective detection methods and pretreatment methods to efficiently separate and enrich various pollutants in complex matrix samples,so as to achieve specific and highly sensitive detection.Based on the high specific affinity of the aptamer for the target analyte,a series of different types of aptamer sensors were designed.Based on the strand displacement reaction,the detection signal of the target was converted into fluorescence or ultraviolet detection signal.Control chip detection signal,gas chromatography detection signal;thus realizing high specificity and high sensitivity detection.In order to save the production cost of the sensor,to ensure the stability of the biosensor,and to avoid the tedious labeling process,a label-free adaptive ligand sensor was selected in this paper.In order to achieve highly sensitive detection and better separation and detection of targets in complex matrix samples,this study combines a series of enzyme-free nucleic acid signal amplification strategies and stirring rod extraction mechanisms.Three types of label-free aptamer sensors were constructed in combination with fluorescence,gas chromatography,and microfluidic chip detection instruments to achieve rapid and sensitive detection of different types of pollutants in milk,fish,and soil.The specific research contents are as follows:1.Zero background and triple-signal amplified fluorescence aptasensor for antibiotics detection in foods.It is important to eliminate matrix interference for accurate detecting antibiotic residues in complex food samples.In this study,we designed a zero-backgrounded fluorescence aptasensor to achieve on-site detection of antibiotic residues,with chloramphenicol(CAP)as representative analyte.Moreover,a three stir-bars assisted target recycling system(TSBTR)was designed to achieve triple signal amplification and increase the sensitivity.The bars included one magnetic stir-bar modified with two kinds of long DNA chains,and two gold stir-bars modified with Y shape-duplex DNA probes respectively.In the presence of CAP,the target could recurrently react with the probes on the bars and replace a large amount of long DNA chains into supernatant.After then,the bars were taken out and SYBR green dye was added to the solution.The dye can specifically intercalate into the duplex structures of DNA chains to emit fluorescence while not emitting a signal in its free state.Under the optimized experimental conditions,a wide linear response range of 5 orders of magnitude from 0.001 ng·mL^-1 to 10 ng·mL^-1 was achieved with a detection limit of 0.033 pg·mL^-1 CAP.The assay was successfully employed to detect CAP in food samples(milk&fish)with consistent results with ELISA's.High selectivity and sensitivity were attributed to the zero background signal and triple signal-amplification strategy.Moreover,the detection time can be shortened to 40min due to that three signal amplified process can occur simultaneously.The fluorescent aptasensor was also label-and enzyme-free.All these ensure the platform to be rapid,cost-effective,easily-used,and is especially appropriate for detection antibiotics in food safety.2.Magnetic stir bars with hyperbranched aptamer as coating for selective,effective headspace extraction of trace polychlorinated biphenyls in soils.It is challenging to greatly increase the extraction selectivity and efficiency of stir bar sorptive extraction of ultra-trace polychlorinated biphenyls(PCBs)in complex environmental matrix,e.g.,soils.To fulfill this purpose,one of the critical works is to prepare some coatings with high selectivity,adsorptive capacity and reusability.It is also important to develop some green,simple methods for building the coatings.In this work,a kind of highly efficient and bioactive coating based on hyperbranched aptamer(HB-Apt)was constructed via hybridization chain reaction(HCR).Then,the HB-Apt was coated on a magnetic stir bar and applied to headspace extraction of PCB72 and PCB106 in soils.The core-shell gold magnetic particles(Fe₃O₄@AuNPs,AuMNPs for short)was employed as the substrate to immobilize the HB-Apt.The extracted PCBs on the stir bar could be easily eluted in ethanol by stirring,and then sampled in gas chromatography-mass spectrometry(GC-MS)for qualification.The ultra-low detection limit(0.003-0.005 ng·g^-1),good linearity(0.01-500 ng·g^-1,R²?0.994)and reproducibility(RSD:4.58-6.53%)were obtained in soils.Compared with the common aptamer coating,the HB-Apt coating not only exhibited good selectivity but also 10 times higher extraction capacity.The results might be related to more aptamer fragments on it than common aptamer to capture the target.The magnetic stir bar can not only be employed for easy headspace extraction,but also facilitate separation and elution.Moreover,the coating could be recycled for at least 60 times before recovery drops below 90%.All these indicated that the assay is simple,robust,environment friendly and promising for detection of trace PCBs in complex environmental samples.3.Dual-mode extraction stir-bar aptasensor based on microfluidic chip for simultaneous detection of multiple food-borne pathogens.Constructing fast,low-cost and convenient food-borne pathogen detection methods is of great significance for ensuring food safety and preventing food poisoning.Therefore,in this study,a portable extraction stir bar was constructed for rapid on-site detection of foodborne pathogens.An extraction stirring rod was prepared by modifying tetramercaptophenylboronic acid on a gold rod,and non-specific adsorption of Vibrio in the sample was performed,followed by reaction in an aptamer solution prepared in advance.The aptamer solution was modified by HRP Streptavidin and biotin-modified aptamers are prepared;after the reaction,the aptamers will specifically capture Vibrio on gold rods to form a sandwich structure.Subsequently,we added a gold bar with a sandwich structure to TMB for display reaction,and realized the qualitative detection of Vibrio by color,then we used a microfluidic chip to quantitatively detect the aptamers eluted from the gold bar.The portable stir bar prepared in this paper realizes dual-mode detection of Vibrio,both real-time and qualitative detection of Vibrio in situ through color changes,and quantitative detection of Vibrio in microfluidic chip changes.This method developed a simple,sensitive,and rapid detection method,which opened the way for the rapid real-time detection of foodborne pathogens.