Study On The Preparation Process Of PAA Targeted Biosensor And Its Application In Detection Of Salmonella In Milk

Foodborne illnesses are caused by the consumption of water or nutrients contaminated with pathogenic microorganisms such as bacteria,fungi,viruses,etc.Among these pathogens,Salmonella infection is considered to be one of the main causes of these disease outbreaks,it has caused high mortality and huge economic losses worldwide,which pose a great threat to human life’s safety and health.However,the existing detection techniques,such as plate count,real-time fluorescent PCR and immunosorbent assay（ELLSA）,are time-consuming,labor-consuming,and weak anti-interference,which cannot meet the needs of rapid and efficient early identification and detection of Salmonella in the field and online.At this time,NMR biosensors with good specificity,high sensitivity and strong anti-interference ability have attracted widespread attention.They can not only achieve rapid and efficient detection,but also have the advantages of portability and so on,which are considered to be a new method with great potential.In this study,we developed a polyacrylate-targeted gadolinium biosensor based on nuclear magnetic resonance（NMR）technology,combining biological immune reaction with ultrafiltration centrifugation,for the rapid detection of Salmonella in milk.Using water-soluble polyacrylic acid with high density carboxyl as the probe skeleton,paramagnetic metal ion Gd³⁺as the magnetic signal source,the high affinity between streptavidin and biotin was used as the bridge to connect with the antibody and the magnetic complex,finally,the target biosensor was obtained.Through the specific binding of antibodies on the sensor to epitopes on the bacterial surface to catch the salmonella in milk.Then use membrane filtration technology to remove the free probes,and finally measure the longitudinal relaxation time of probes in the collected filtrate with nuclear magnetic resonance.Finally,the longitudinal relaxation time（T₁）of the probe was measured by NMR.The main research contents are as follows:1、Study on the design and preparation process of polymer polyacrylic acid targeting biosensor:The polymer water-soluble polyacrylic acid（PAA）was selected as the skeleton of the whole probe,and the paramagnetic metal nanoparticle Gd was used as the magnetic signal source.EDC/NHSS was introduced to react with polyacrylic acid to activate the carboxyl group to improve the coupling efficiency,and streptavidin（SA）and Gd³⁺were combined to the skeleton PAA step by step.Using the high affinity between streptavidin（SA）and biotin（Bio）as a bridge,SA-PAA-Gd was coupled with a biotinylated monoclonal Antibody（Bio-antibody）to obtain the target probe.Optimize the synthesis conditions of the complex SA-PAA-Gd.The results show that the optimal conditions of the SA and PAA reactions are the mass ratio of 30μg,the reaction time of 3 h,PAA and Gd³⁺ion reactions,a p H of 3.5,and 1h reaction at 25℃.The successful synthesis of SA-PAA-Gd is determined through Zeta potential,Fourier infrared spectrum,X-ray optoelectronic energy spectrum（XPS）.The antibody activity in Bio-Antibody was analyzed by SA-HRP enzyme ratio method to verify the successful synthesis of Bio-Antibody.In addition,using the inductive-induced plasma launch spectrum（ICP-OES）to determine the magnetic complex SA-PAA-Gd,the longitudinal relaxation time（T₁）of SA-PAA-Gd was measured under different Gd³⁺concentration conditions,thereby determine the vertical relaxation rate of the magnetic complex.2、Construction of NMR detection system.In order to achieve rapid and efficient testing of target bacteria,the amount of Bio-Antibody in the detection system,the amount of the complex SA-PAA-Gd,the time of the probe capture Salmonella,the temperature,etc.have been optimized in detail.The optimization results showed that the optimal dosage of magnetic complex SA-PAA-Gd was 120μL,the optimal amount of Bio-Antibody was 20μg,the optimal capture time of Salmonella was 60 min,and the optimal capture temperature was 37℃.Under the circumstances,the NMR detection system signal output was the most stable,the optimal performance evaluation results can be achieved,and the obtained probe capture effect is the best.3、Performance evaluation of NMR Biosensor and its application to detection of Salmonella in milk.In order to ensure the detection effect of the actual milk sample,the performance evaluation of the design synthetic biosensor needs to be performed.It mainly conducts detailed experimental analysis from specificity,anti-interference,detection of sensitivity in PBS solution and cow milk samples,and reproducibility.The research results show that the sensor has good performance advantages in the specificity and anti-interference tests of introducing 1,3 and 5 kinds of non-target bacteria,in the meantime,the detection sensitivity in PBS solution and milk samples has reached 3.3×10³CFU/m L.In addition,the biosensor also has good reproducibility for the detection of Salmonella in milk,and the RSD of the detection result is 2.98%.Therefore,we can determine that the PAA-targeted biosensor designed in this study has great application advantages in the detection of Salmonella.Its strong anti-interference property has a high application prospect in the composition of complex,diverse matrix sample detection,and it also has certain guiding significance for the detection of other pathogenic bacteria that may appear in food.