Construction Of Aptamer Coding Probe-based Microfluidic Chip For Simultaneous Detection Of Multiclass Contaminants

In recent years,food safety and hygiene issues have frequently arisen during the production and processing of food.Contaminants in food often coexist in multiple categories,which will cause direct or potential harm to human health.Common food contaminants are chemical contaminants,including veterinary drugs,pesticide residues,and mycotoxins;and biological contaminants,including foodborne pathogens such as Vibrio parahaemolyticus and Salmonella.Therefore,it is great significance to develop a series of methods that can quickly and simultaneously screen multiclass contaminants in food for ensuring people’s quality of life and safety.However,current food contaminant detection methods,such as chromatography methods and immunology methods,have some problems such as long detection time,high detection cost,complex sample pre-treatment,and low sensitivity,which are difficult to realize the portability,rapid and low cost requirements of food safety monitoring.Microfluidic chip has attracted great attention and favor in the field of analysis and separation in recent years due to the characteristics of fast response,portable,low analysis cost and high throughput.In this paper,we proposed an aptamer coding probe-based microfluidic chip platform,combined with magnetic separation technology and rolling circle amplification（RCA）signal amplification strategy to develop a series of rapid,high-sensitivity,high-throughput,high-specific and low-cost methods for simultaneous detection of multiclass contaminants in food.The main contents of this study are as follows:1.A microfluidic chip based ratiometric aptasensor for antibiotic detection in foods using stir bar assisted sorptive extraction and rolling circle amplificationA ratiometric and sensitive microfluidic chip based aptasensor was developed for antibiotic detection,with kanamycin（Kana）as model analyte.A novel stir bar assisted sorptive extraction and rolling circle amplification strategy was designed to largely amplify the signal and overcome complex matrix interference in food samples.The detection mechanism was as following:firstly,many duplex DNA probes（a single-stranded DNA as primer hybrid with aptamer sequence）were modified on a stir bar.In the presence of Kana,the probes on the bar could specifically capture Kana and release primer to trigger RCA in the presence of circular DNA template（CDT）.With the reaction proceeds,the amount of CDT decreased and RCA products increased.It is worth mentioning that they can be efficiently separated and detected by microfluidic chip.The signal ratio of RCA products and CDT（I_R/I_C）can be employed to qualify Kana in a wide linear range from 0.8 pg·mL^-1to 10 ng·mL^-1with low detection limit of 0.3 pg·mL^-1.This method exhibited excellent sensitivity,selectivity and can obviously reduce matrix interference through ratiometric strategy combing with stir bar extraction.The aptasensor was successfully tested in milk and fish samples,confirming that it can be applied for on-site quantitation of antibiotic residues in foods.2.Simultaneously responsive microfluidic-chip aptasensor for determination of Kanamycin,aflatoxin M1 and 17β-estradiol based on magnetic tripartite DNA assembly Nanostructure probesThe authors describe a microfluidic chip-based aptasensor platform combined with magnetic tripartite DNA structure-functionalized nanocomposites to achieve simultaneous determination of Kanamycin（KANA）,aflatoxin M1（AFM1）,and 17β-estradiol（E2）in milk.First,the two-duplex tripartite DNA Nanostructure was first assembled on the surface of magnetic beads.When the aptamer on the probes recognized the specific target,the aptamer-target would be released into the supernatant.The pre-primer@circular DNA template structure initiates rolling circle amplification（RCA）by phi 29 polymerase.After magnetic separation,the magnetic nanocomposites were added into a solution containing three different lengths of complementary strands to the RCA products.The number of complementary strands would dramatically decrease,and be quantitated by microfluidic chip.Further,the employment of magnetic nanocomposites and microfluidic chip not only resolve the complex matrix interference,but also dramatically enhance the determination selectivity and sensitivity.This aptasensor allows for determination of KANA,AFM1,and E2 with limits of detection as low as 0.32 pg·mL^-1,0.95 pg·mL^-1,and 6.8 pg·mL^-1,respectively.This novel method exhibits the advantages of excellent stability and fast simultaneously responsive time（<3 min on microfluidic-chip platform）for determination of KANA,AFM1,and E2 in milk samples and ensure food safety.3.Magnetic Nanoprobe-based colorimetric and microfluidic-chip dual-mode aptasensor for simultaneous determination of Vibrio parahemolyticus and Salmonella typhimuriumFoodborne diseases caused by microorganisms have increasingly become worldwide attention.Herein,we developed a sensitive and point-of-care colorimetric and microfluidic chip dual-mode aptasensor combined with magnetic Nanoprobe for simultaneous determination of Vibrio parahemolyticus and Salmonella typhimurium.Colorimetric aptasensor was established first for detecting the ATP in bacteria due to bacterial ATP detection allows to verify microbial contamination.The rolling circle amplification products（RP）-conjugated magnetic composite capture probes（RMNPs）were used as capture probes,and the G4 sequence could catalyze the oxidation of TMB,resulting in the visible color change by naked eye,without relying on any complex instruments,can reflect the concentration of bacteria.After verifying bacterial contamination in food,the accurate quantification of food-borne S.typhimurium and V.parahaemolyticus was further analyzed with microfluidic chip platform.RMNPs and Eco RV endonuclease strategies were employed as the signal capture,amplification and discrimination element.The results indicate that the proposed colorimetric and microfluidic chip dual-mode aptasensor is a promising for point-of-care qualitative detection of bacteria contamination and multiplex foodborne pathogenic bacteria simultaneous detection in food.