| Food biological hazards represented by foodborne pathogens and mycotoxins are important factors threatening food hygiene and safety in China.However,the diversity of food types,the complexity of the ingredients and the timeliness of production and transportation have made rapid and accurate quantitative detection become the research difficulty of food biological hazard control.At present,bacterial culture and liquid chromatography-electrospray mass spectrometry（LC-MS）as the“gold standard”of food detection has the disadvantages of poor timeliness,cumbersome operation and high cost,which is difficult to meet the requirements of food regulatory authorities.Microfluidic systems based on Micro Electro-Mechanical System（MEMS）have received widespread attention in the field of rapid detection due to their miniaturization,integration and automation advantages.In addition,the closed structure of microfluidic chips and automated processes effectively reduce the risk of contamination caused by manual operation.Currently,microfluidic-based rapid detection systems are an effective means to address global food safety challenges by integrating biomolecular detection methods into microfluidic chips to develop miniaturized and cost-effective analytical platforms.According to the actual needs of food safety,this paper organically combines microfluidic technology,novel molecular biology detection methods based on specific molecular recognition elements and signal transduction mechanisms of nanomaterials to establish a series of highly sensitive,high-throughput and stable microfluidic detection platforms for foodborne pathogenic factors,forming a system for controlling and accurately tracing common biological hazards from food.The main research contents are as follows:1.In order to establish a universal scheme for highly accurate detection of foodborne pathogens,a high-fidelity target microfluidic identification（HFTMI）strategy based on pan-genomic analysis and digital nucleic acid detection technology was established,and a proof-of-concept study for rapid detection of foodborne pathogens was conducted using Vibrio parahaemolyticus as a model.A local database consisting of the genome sequences of 872Vibrio parahaemolyticus strains and 743 non-Vibrio parahaemolyticus strains in the NCBI public database was analyzed by a pan-genomic analysis method established by the team.23specific molecular targets of Vibrio parahaemolyticus were identified,and group＿41170 with high sensitivity and broad spectrum was screened as the high-fidelity target of Vibrio parahaemolyticus.The partitioning efficiency of the q RAA reaction on a chamber-based digital microfluidic chip was improved by investigating different power systems.Compared to q PCR,the HFTMI strategy exhibited higher sensitivity and tolerance in detecting real samples with a minimum quantifiable concentration of 102 CFU·m L-1 for artificially contaminated samples.In addition,HFTMI has the advantages of simple operation,low equipment complexity,lower time cost than commercial droplet digital PCR,and high detection capability for various types of samples.The HFTMI strategy can provide strong support for the development of absolute quantitative detection methods for different pathogens and provide more reliable guidance for food safety.2.Due to the limitation of digital chip in flux,a high-throughput centrifugal microfluidic chip based on real-time fluorescence was developed in this study to meet the actual demand of simultaneous detection of multiple pathogenic microorganisms in food safety supervision.In order to achieve accurate and rapid traceability of Salmonella serogroups in food,the SSG-RAA HTM high-throughput microfluidic system was developed to simultaneously detect Salmonella spp.and 5 serogroups（B,C1,C2-C3,D,E）based on the Salmonella serogroup-specific molecular targets and real-time fluorescent recombinant enzyme-assisted amplification（q RAA）that our team has mined.The SSG-RAA HTM system offers significant advantages in terms of detection time（15～40 min）and reliability of result interpretation compared to conventional slide agglutination methods.This system has a LOD of 101 CFU·m L-1（genus and serogroups B,C1,C2-C3）or 102 CFU·m L-1（serogroups D,E）.The system has good stability（C.V.s<5%）and accuracy（>98%）in actual sample validation.3.Based on the above centrifugal microfluidic platform,the specific targets of 11Salmonella serotypes mined by the team in the early stage were used as molecular recognition elements to establish the corresponding programmable LAMP system（LAMP-chip）to achieve rapid and accurate identification of common Salmonella serotypes in Chinese retail food.The programmable LAMP developed with Au NPs as the carriers of single-stranded DNA primers and polymerase can effectively suppress false positives caused by mismatches and low-temperature false annealing.Moreover,LAMP-chip exhibit good stability and interference resistance.The genetic serotyping strategy based on the programmable LAMP can complete the entire detection process within 40 min with a detection limit of 102 or 103 CFU·m L-1.The accuracy of the strategy was 100%for 688 Salmonella and 22 non-Salmonella strains with reference to standard culture and identification methods.The LAMP-chip showed good recovery in the detection of real samples with a precision of 85.9%～105.2%with RSD<10%.Compared to the slide agglutination method,which can only identify a single serotype,the LAMP-chip can achieve the simultaneous detection of multiple Salmonella serotypes in food.In addition,the genetic serotyping strategy allows the detection module on the chip to be modified according to the trend of the most popular serotypes to meet the latest needs of the food safety related departments for Salmonella surveillance.4.In order to simplify the operation process of competitive immunometric assay（CIMA）and realize the rapid and sensitive detection of small molecular mycotoxins,a polydimethylsiloxane gravity-driven circulating microfluidic chip based on dual-signal mode was developed to realize CIMA automation through modular design and used for the detection of aflatoxin B1（AFB1）.The structural design of the chip,together with the two-wavelength quantum dot ratio fluorescence,effectively eliminates the influence of environmental factors,improves the signal stability,and ensures that the final detection result positively correlates with the target concentration.Moreover,the theoretical analysis performed for the established physical model of the three-dimensional reaction interface inside the chip confirmed the improved reaction rate of immune adsorption in the microfluidic strategy.Overall,the method exhibited a wide analytic range(0.2～500 ng·m L-1),low detection limit(0.06 ng·m L-1),high specificity,good precision（coefficient of variation<5%）,excellent reusability（20 times,89.1%）and satisfactory practical sample analysis capacity.Furthermore,the reusability and designability of this chip provide a reliable scheme for situ detection of AFB1 and can be applied to the multivariate visualization of multiple mycotoxins and foodborne pathogens and to achieve rapid quantitative detection of low-abundance targets.5.Limited by the principle of CIMA,the excessive modular design on the microfluidic chip makes it difficult to improve the detection flux through array.Therefore,developing an integrated small molecule detection method suitable for parallel distribution on chip is an effective strategy to realize the high-throughput detection of mycotoxins.Based on the recognition sequences of six mycotoxins,trigger switches of CRISPR system were constructed and a portable high-throughput microfluidic hypersensitive detection platform（FMB system）was established for the precise detection of six hazardous mycotoxins in food by the signal transduction and enhancement mechanism of quantum dots and photonic crystals.The method used DNA sequences with specific recognition functions to construct trigger switches containing activators and simulates the secondary structure by Mfold,and analyzes the feasibility of the trigger switches by polyacrylamide gel electrophoresis and real-time fluorescence curves.A universal quantum dot probe of CRISPR system and a photonic crystal substrate with matching photonic band gaps were constructed to achieve a significant enhancement of the output signal up to 45.6-folds.The transition-state CRISPR system was constructed by adjusting the ratio of cr RNA and activator sequences composition to achieve high-response to low concentrations of target mycotoxins.The results showed that each detection unit in the FMB system had good specificity and the detection limit could reach fg·m L-1.The FBM system with lyophilized CRISPR-related reagents still had ultra-sensitive analytical performance and good stability.The practicality of the FMB system was evaluated using actual samples,and all results were in high agreement（88.76%～109.99%）with the HPLC method.In summary,this thesis developed a series of microfluidic detection platforms for high-throughput and hypersensitive detection of foodborne pathogenic factors.For foodborne pathogens,a high-fidelity target microfluidic identification strategy was constructed to achieve absolute quantitative detection of pathogens by using Vibrio parahaemolyticus as a research model.In addition,based on the mined molecular targets of Salmonella serogroups and serotypes,high-throughput genoserotyping system was established on centrifugal microfluidic chips using q RAA and programmable LAMP respectively.These strategies have achieved multiple target identification in the same sample,and further improved the reliability and accuracy of Salmonella screening results.For foodborne mycotoxins,an integrated gravity-driven CIMA chip with dual-signal mode strategy for portable detection of a single mycotoxin AFB1 and an ultrasensitive microfluidic biosensor based on functional DNA regulation and transition-state CRISPR system were established for high-throughput detection of mycotoxins.These strategies enable highly integrated molecular detection methods through efficient rational design of microfluidic chips,providing theoretical and technical support for the development of high-throughput ultrasensitive microfluidic analysis systems for risk identification,traceability and rapid detection of foodborne pathogenic factors. |
