The Construction Of CRISPR/Cas12a Responsive Electrochemiluminescence Sensors And Their Applications In Foodborne Pathogen Detection

Foodborne pathogen can be transmitted to humans through food,and then cause food poisoning.Swift and responsive detection of foodborne pathogen is the most important method to hinder and control foodborne pathogen infection.However,the existing detection methods still cannot meet this demand.Therefore,developing a fast and sensitive detection method is an urgent problem to be solved at this stage.ECL combined with CRISPR is used to construct a biosensor to realize sensitive and efficient detection of foodborne pathogen.Using logic gate to detect two kinds of foodborne pathogen simultaneously greatly improves the detection flux and provides a novel tool for foodborne pathogen detection and the early diagnosis after infection with foodborne pathogen.The sensor showed high sensitivity to the detection results of foodborne pathogen in actual samples,and it is expected that this method will have a broad potential application for food safety and biosensing.1.Allosteric probe-triggered isothermal amplification to activate CRISPR/Cas12a for sensitive electrochemiluminescence detection of SalmonellaWe propose a sensitive ECL platform for the detection of Salmonella by employing CRISPR/Cas12a system activated by allosteric probe-triggered isothermal amplification.In the presence of Salmonella,the structure switching occurs on allosteric probes,resulting in their recognition and hybridization with primers to trigger isothermal amplification.Salmonella is then released to initiate the next reaction cycle accompanying by generating a large amount of ds DNA,which is subsequently recognized by cr RNA for activating the trans-cleavage activity of Cas12a.Furthermore,the activated Cas12a can indiscriminately cut the ss DNA which is bound to the electrode,enabling the release of the ECL emitter porphyrinic Zr metal-organic framework(MOF,PCN-224)and exhibiting a decreased ECL signal accordingly.The detection limit of the sensor is down to 37 CFU/mL,and showing a superior linear correlation between 50 CFU/mL and 5×10~6 CFU/mL.The sensor can quickly and sensitively detect Salmonella in different types of actual samples with good specificity and sensitivity.2.ECL sensor for simultaneous detection of Salmonella and S.aureus via using logic gate double signalsIn order to increase the detection throughput,based on the previous chapter,this chapter constructs an ECL sensor platform to detect both Salmonella and S.aureus.This method uses highly specific aptamer to identify the target,and isothermal amplification is combined with Cas12a with single base resolution to amplify the signal,which improves the sensitivity and specificity of the biosensor.When S.aureus occur,isothermal amplification is triggered by the release of blocking chains,and ds DNA is formed by the combination of product DNA with ss DNA on the electrode.After CV embedding,ECL signal of La-MOF is quenched.The allosteric probe structure changes when Salmonella occurs,triggering isothermal amplification,which in turn activates the trans-cleavage activity of Cas12a.Activated Cas12a can lower the ECL signal by cutting the ss DNA that binds to the electrode,causing Ru@DMSNs to fall off the electrode surface.The detection limit of S.aureus is down to 11 CFU/mL,showing a wonderful linear correlation between50 CFU/mL and 5×10~6 CFU/mL.The detection limit of Salmonella is down to 27 CFU/mL,showing an excellent linear correlation between 50 CFU/mL and 5×10~6 CFU/mL.The biosensor shows excellent performance in the detection of foodborne pathogen and offers a novel technique for food safety detection.