| Foodborne pathogenic bacteria contamination caused by Staphylococcus aureus(S.aureus)seriously endangers human health.However,traditional bacterial detection methods have obvious disadvantages,such as plate separation method,which is usually time-consuming and complex;The enzyme-linked immunosorbent assay(ELISA)method has a low limit of detection.Although polymerase chain reaction(PCR)is a simple,convenient and sensitive method for bacterial detection,it is easily affected by food matrix and bacterial concentration,and there are certain limitations for the detection of larger volume food samples.Traditional magnetic separation techniques are considered an efficient and rapid pretreatment protocol in trials,for example in the rapid detection of pathogenic bacteria,which perform well.However,the high price,cumbersome preparation process and high storage cost of antibodies lead to the inability of magnetic separation technology to be widely used in practical detection.Vancomycin(Van)is a glycopeptide antibiotic that inhibits bacterial proliferation by blocking cell wall synthesis,thereby inhibiting bacterial infection and exhibiting strong antibacterial effects against gram-positive bacteria such as S.aureus.However,studies have shown that a small amount of Van does not affect the vitality of gram-positive bacteria for a short period of time,but can interact with the terminal D-ala-d-alanine(D-ala-d-ala)on the cell wall of gram-positive bacteria through the heptapeptide on the skeleton.The binding force of Van to target bacteria is similar to the antigen-antibody binding force,and based on this,it has been widely used in recent years to isolate and detect recognition molecules of gram-positive bacteria.In this paper,Van was used as the identifying molecule,combined with different"molecular arms"to modify carbon nanotubes,and dispersed in simulated liquid or food samples to form a three-dimensional interwoven network structure for the isolation and enrichment of S.aureus,and the isolation and enrichment behavior of the network on S.aureus in simulated solution and fresh milk was studied.The results show that the antibacterial activity of carbon nanotubes is imparted by grafting modification,and the dispersion of carbon nanotubes in the solution system is improved,so that they can be uniformly suspended in solution,and S.aureus can be efficiently separated and enriched,and even low-level target bacteria can be isolated and enriched.This study demonstrates the potential application value of the modified complex in the rapid and efficient detection of S.aureus.Compared with magnetic separation research,it provides a new way for the isolation and enrichment separation of foodborne pathogenic bacteria without the help of external magnetic field.Furthermore,the effect of three different"molecular arms"vectors on the capture efficiency of S.aureus was analyzed.The research is as follows:1)A three-dimensional interwoven network composed of vancomycin-polyethylene glycol diamine-modified carbon nanotubes(Van-PEG-MWCNTs)was constructed for the isolation and enrichment of S.aureus.The isolation and enrichment behavior of the network on S.aureus in simulated solution and real milk was studied.Grafted vancomycin-polyethylene glycol diamine(Van-PEG)on carbon nanotubes confers the activity of carbon nanotubes against S.aureus,and at the same time improves the dispersion of carbon nanotubes in the solution system,so that they are uniformly suspended in the solution to form a loofah-like three-dimensional structure,so that the grafted complex can efficiently separate and enrich S.aureus in the solution system,and even isolate and enrich low-level target bacteria.The results showed that Van-PEG-MWCNTs,as molecular probes,were able to identify,isolate and enrich S.aureus from phosphate-buffered saline solutions and fresh milk samples with LOD of 2×10~1 CFU/m L.This study demonstrates the potential application value of Van-PEG-MWCNTs in the rapid and efficient detection of S.aureus.2)Van and CS-modified functionalized MWCNTs were prepared by carbodiimide coupling,and a method was successfully constructed to obtain Van-CS-MWCNTs by using CS as rocker arm to modify carboxylated MWCNTs and then grafted Van.The effects of thermal stability,specificity and multi-binding site"molecular arm"modification of Van-CS-MWCNTs on the capture rate of S.aureus were studied.The results showed that the use of CS with poor hydrophilicity as the"molecular arm",although the grafting amount of Van was increased,the dispersibility of Van-CS-MWCNTs was poor,and the capture rate was lower than that of Van-PEG-MWCNTs on S.aureus.Compared to pure MWCNTs,only a slight advantage is shown.3)A method was constructed to obtain Van-COS-MWCNTs composites by modifying carboxylated MWCNTs with COS as the rocker arm and then grafting Van.Van-COS-MWCNTs are used as molecular probes to isolate and enrich S.aureus from PBS solutions,raw milk samples,and purified water.Universal recognition molecules are used to specifically identify S.aureus,where COS acts as a"molecular arm"that increases the amount of Van grafting,thereby increasing contact with the D-Ala-D-Ala molecule on the surface of S.aureus.Under optimized conditions,the Van-COS-MWCNTs complex constructed here was used in a 10 m L system:even with bacterial concentrations as high as 2×10~7CFU/m L,the capture efficiency was higher than 90%in PBS solution and purified water matrix,and the capture efficiency in raw milk was higher than 83%.Moreover,the capture effect of the long molecular chain"COS"with good hydrophilicity and large number of amino groups as the carrier of the molecular arm was better than that of"CS"with a large number of amino groups but poor hydrophilicity,and"PEG"with better hydrophilic effect and only two amino groups at both ends.This method demonstrates the potential application value of Van-COS-MWCNTs carrier with COS as the"molecular arm"for the rapid and efficient separation and enrichment of high concentrations of S.aureus in food sample matrix. |
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