Rapid Detection Of Three Food Hazards By Colorimetric Sensing Methods Based On Stimuli-responsive Polydiacetylene

The incidence of food-borne diseases caused by exposure to,or intake of various allergens,microbiological,chemicals,or natural toxins is increasing yearly,resulting in serious public health problems and huge financial losses worldwide.Hence,it is extremely important to establish rapid detection systems targeting food hazards,that can be employed on the spot for fast results.Such rapid screening assays often rely on colorimetric detection systems.In recent years,a promising strategy towards colorimetric screening has relied on the use of polymer polydiacetylene（PDA）.Due to its unique molecular structure and physicochemical properties,the stimuli-responsive PDA can produce a blue-to-red color transition,when it is stimulated by an external stimulus.This property makes it very attractive for fast colorimetric sensing.However,when it comes to food safety screening,there are still some limitations of colorimetric sensing based on PDA.These limitations include the complicated operational procedure,insufficient sensitivity,non-universal applicability,and the poor traceability of the detection data.These drawbacks pose great challenges to the development of PDA-based methods for food safety inspections.To overcome the limitations,in this work,we investigated the colorimetric properties of PDA for different colorimetric sensing systems by preparing and characterizing functionalized PDA.Next,PDA was successfully applied in the detection of three typical food hazards in the categories of allergens,microbiological and chemicals,by designing different sensing strategies.Finaly,the PDA-loaded colorimetric strip tests were developed and thoroughly studied towards the colorimetric detection of histamine and monitoring of fish food spoilage.After that,to improve the specificity and sensitivity of the detection method,loop-mediated isothermal amplification（LAMP）and immuno-chemical systems were introduced to combine their selectivity with the convenience of PDA-based detection,simultaneously achieving the amplification of the colorimetric signal for the detection of antibiotic-resistant Staphylococcus aureus and diethylstilbestrol（DES）,respectively.Finally,in all the above detection methods,a smartphone was used to analyze the colorimetric information,to record and trace the detection results,thus facilitating the establishment of a more intelligent and interconnected food safety monitoring system.The research contents are as follows:1.To explore the potential application for PDA-based colorimetric sensing,the preparation and optimization process of PDA as a colorimetric probe was investigated,and its performance against different external stimuli was preliminarily evaluated.First,the synthetic procedure to produce PDA vesicles was optimized by experimenting with different monomers and their molar proportions,as well as the reaction time and the related organic solvent.Next,to investigate the application of PDA for specific biosensing,by using the optimized conditions,antibody,streptavidin,and nucleic acid functionalized modified PDA were successfully prepared and characterized.Not only did we investigate its colorimetric responses to temperature,p H,formation of immuno-complexes and nucleic acid-complexes,but we also confirmed this by using different characterization methods,including UV spectrophotometry,contact angle measurements,Raman spectroscopy and electrochemical analysis.Finally,the results indicated the prepared PDA had good colorimetric response,and SA,antibodies and nucleic acids could be successfully modified on the surface of PDA.In addition,the responsiveness,stability,and reproducibility of PDA in both liquid-and paper-based systems was good,which means that PDA is a promising material for application in the colorimetric detection of food hazards.2.Based on the above research on PDA characteristics,PDA was used here as the colorimetric strip to simple and rapid detection method for histamine in spoiled fish in combination with a smartphone and,which overcoming the limitations of histamine detection methods regarding the reliance on precision instruments and the inability to report results in a timely manner.After analyzing the colorimetric properties of PDA,optimizing the colorimetric membranes,the buffer,and the incubation time of the test strip,a histamine colorimetric test strip with PDA as p H indicator was successfully prepared.Then,the quantitative detection was further achieved by analyzing the gray value of the test strips using the Color Grab application on a smartphone and the open-source software Image J,with good reproducibility and sensitivity in the concentration range of 70～2240μg/m L,with a visual detection limit of 70μg/m L.Finally,the developed devices were successfully integrated into packaging and used to monitor the spoilage of canned fish.3.While successfully established for testing the histamine,PDA by itself is not a selective colorimetric label.Therefore,to enhance the specificity and sensitivity,PDA-based detection was combined with the sensitive and highly specific LAMP technology,to realize the on-site colorimetric detection of antibiotic-resistant Staphylococcus aureus in milk.First,through metagenomic sequencing of raw milk,cow manure,river water and aerosol deposit of dairy farms around Tianjin,the most abundant antibiotic resistance gene was identified as the mac B gene.Meanwhile,the SAOUHSC＿01275gene was screened as a specific housekeeping gene of Staphylococcus aureus,which can be used as a marker for its presence.Then the LAMP assays were established for the above two target genes.By comparing different LAMP color indicators（PDA,phenol red,and a commercial kit）,it was finally confirmed that PDA shows the best colorimetric properties to detect LAMP.In addition,semi-quantitative detection was achieved by collecting the RGB information of the LAMP-PDA system through use of smartphone-based detection.When the concentration of genomic DNA between9.248×10^-7 ng/μL and 9.248×10^-3 ng/μL,the colorimetric intensity is linear with the log10 value of the concentration of genomic DNA（R²=0.9846）with a visual LOD of 9.248×10^-7 ng/μL.Finally,using lyophilized LAMP reagents and portable equipment,the LAMP-PDA system was successfully used to confirm antibiotic-resistant Staphylococcus aureus on dairy farms within 1 hour after sample acquisition.Most importantly,the LAMP-PDA assay is universal approach,and by changing the target gene,it can be extended to detect any other antibiotic resistance genes and other bacteria.Moreover,the developed workflow is well suited for resource-constrained settings.4.Here,another application of targeting small molecule diethylstilbestrol（DES）was explored to match the excellent chromogenic properties of PDA with selective biorecognition,namely by functionalization to antibodies.Two signal-enhanced immune strategies were constructed and portable colorimetric detection for DES was realized in both a homogeneous state（solution）and a paper-based state after exploring the mechanism of PDA aggregation behavior on the color response.First,a bifunctional ligand,biotin-modified diethylstilbestrol（Bio-DES）was synthesized as a functional bridge.Next,streptavidin functionalized PDA（PDA-SA,blue color）and DES antibody functionalized PDA（PDA-DES antibody,blue color）were synthesized.By designing different immune-sensing strategies,under optimal conditions,the aggregated PDA immunocomplex（red color）was obtained through the biotin-streptavidin system.When DES was present in the analyte,the aggregation of PDA was inhibited due to the competitive immunoassay,and the resulting color of the solution would be blue.After analyzing the color information by Color Grab,a good linear relationship（R²=0.9901）between the color change and the logarithm of DES concentration was obtained when the DES concentration ranged from 2 to 1250 ng/m L,with a smartphone detection limit of 0.85 ng/m L and visual detection limit of 10ng/m L.In addition,it was verified that the homogeneous and paper-based methods have good selectivity and accuracy,which means that it is applicable for the on-site detection of DES.It is worth noting that this assay can also be extended to the detection of other targets by simply designing the bifunctional ligand and choosing appropriate antibodies.To sum up,in this thesis,three PDA-based colorimetric sensing systems have been established and successfully applied for rapid detections of histamine,antibiotic resistance gene and DES in food samples.The detection strategies designed in this study broaden the ideas for the detection of food hazard factors based on PDA colorimetric sensing and provide a research basis for the construction of more portable,sensitive,and robust immune/nucleic acid sensing methods based on PDA,demonstrating important theoretical value and application prospects.