Construction Of Glycan Sensing Interface And Its Application For Pathogenic Bacteria-Mannose Interaction Study

The study on selective recognition between pathogenic bacteria and glycan can provide new solutions for the pathogenic bacteria isolation,enrichment,detection,identification,biofilm inhibition and screening of antibiotic alternative drugs.However,the lack of rapid,sensitive and reliable detection techniques and methods is an important reason for restricting its development.Glycan biosensors have shown their good development prospects in the study of the interaction between pathogenic bacteria and glycans with their unique advantages.The current detection technologies applied for investigating interaction between pathogenic and glycan rely on large instruments and need for fluorescent labeling.In this study,it is proposed to investigate interaction between pathogenic bacteria and glycan using electrochemical impedance biosensors which are fast,sensitive,label-free and easy to integrate and miniaturize.Combining with the isothermal adsorption model,a novel method was established for quantitatively calculating the binding affinity between pathogenic bacteria and glycan.Sensing interface is a key component of the glycan biosensor.In order to overcome the problems of weak affinity and poor selectivity between pathogenic bacteria and glycan in vitro,a nano-glycan sensing interface and a biomimic glycoprotein interface were constructed.A glycan biosensor can simultaneously obtain a variety of effective information by combining with Electrochemical Impedance Spectroscopy?EIS?and Surface Enhanced Raman Spectroscopy?SERS?dual detection mode.It can be used to study the interaction between pathogenic bacteria and glycan and identify different pathogenic bacteria expressing type 1 pili.Based on the selective recognition of pathogenic bacteria and glycan,the combination of glycan biosensor,glycosylated magnetic microspheres and microfluidic chip provides a new method for rapid and effective evaluation of the efficacy of carbohydrate anti-adhesion reagents and detection of Salmonella.The main research work and results of this paper include:?1?The Man/MUA-MH/Au sensing interface was constructed to study the interaction between pathogenic bacteria and mannose.A new method based on impedance detection for pathogen-glycan interaction studies was developed.Self-assembly and covalently bonded methods were used to construct a bioactive and well-stabilized Man/MUA-MH/Au biosensing interface.Through the impedance detection and Randless equivalent circuit model analysis,the corresponding electron transfer impedance(R_et)owing to pathogenic bacteria captured on the mannose sensing interface was obtained.According to the Frumkin isotherm model,the binding affinity between bacteria and mannose was calculated.The interaction between mannose on the sensing interface of Salmonella typhimurium ATCC14028 and Escherichia coli JM109 was studied by this method.The results showed that the affinity of mannose on the sensing interface between the two pathogens were K_ADS?S.T.?=2.16×10⁶?CFU/mL?^-1 and K_ADS?JM109?=5.84×10³?CFU/mL?^-1 respectively.It indicates that the sensing interface has strong selective adsorption capacity for S.typhimurium ATCC14028,and also has certain adsorption capacity for E.coli JM109 which express type 1 pili.This method provides a simple,rapid and reliable way for the quantitative study of the interaction between bacteria/cells and biomolecules on the interface.?2?Construction of Man/MUA-MH/Au NPs nano-sensing interface and pathogen-gly-can interaction study by dual-mode detection.Au NPs?Gold nanoparticles?were prepared on the surface of clean gold electrode by potentiostatic deposition.Then,the Man/MUA-MH/Au NPs composite nano-sensing interface was prepared by chemical self-assembly.The interaction between the interface and S.typhimurium ATCC14028,E.coli JM109,E.coli DH5?,Staphylococcus aureus and Pseudomonas aeruginosa were investigated by EIS.The results showed that the glycan interface Man/MUA-MH/AuNPs selectively capture S.typhimurium ATCC14028 and E.coli JM109 that express type 1 pili?containing FimH adhesin?,and the binding affinities were 6.859×10²³ M^-1 and 2.054×10¹⁷ M^-1 respectively,without capture other bacteria.Furthermore,the SERS effect of nano-gold on the composite nano-sensing interface was used to obtain the SERS spectrum of the pathogenic bacteria captured on the interface in situ,and the partial least squares-discriminant analysis?PLS-DA?was used to identify S.typhimurium ATCC14028 and E.coli JM109.This method solved the problem that current glycan biosensors cannot distinguish between different bacteria that can be selectively recognized by the same glycan.?3?Based on the construction of the nano neoglycoprotein sensing interface,an integr-ated impedance sensing bacterial chip was developed to carry out the efficacy test of anti-bacterial adhesion reagent.S.typhimurium and E.coli?type 1 pili?are selectively recognized by glycoproteins on the surface of host cells rather than free cabohydrate.Therefore,it is proposed to construct Man-BSA interface at the nanoscale to better simulate the presentation and arrangement of cell surface glycans and promote the occurrence of multi-valency interactions.A microfluidic chip integrated with inlet zone,mixing zone,buffer zone,detection zone and outlet zone was designed and prepared.Four microelectrodes with new structure integrated in the detection zone,and Man-BSA@Au NPs nano neoglycoprotein interface was construct on the working electrode.The microfluidic chip was used to evaluate the anti-adhesive effect of S.typhimurium anti-adhesion reagent.The results showed that the interface can selectively capture S.typhimurium,while The mannoside reagent D-Man,Me-Man,Phenyl-Man,Mannatide can inhibit the adhesion of S.typhimurium to Man-BSA@Au NPs.The anti-adhesion efficiency was Phenyl-Man,Mannatide,Me-Man,D-Man in turn,which were consistent with the traditional methods in the literature,indicating that the construction of a biomimetic interface in a specific functional area on a microfluidic chip can effectively evaluate the anti-adhesion effect of carbohydrate compounds.This study provides a simple,economical,rapid method for the evaluation of the efficacy of anti-adhesion drugs for pathogenic bacteria.?4?Based on the selective recongnization of Salmonella and E.coli by mannose,a new method for the detection of pathogenic bacteria was developed.The detection performance of S.typhimurium by electrochemical impedance biosensor based on Man/MUA-MH/Au,Man/MUA-MH/Au NPs and Man-BSA@Au NPs three mannose sensing interfaces were studied.The detection limits were 50CFU/mL,10 CFU/mL,and 5 CFU/mL respectively.The detection method based on the Man-BSA@Au NPs interface and the integrated impedance biosensor bacterial chip exhibits excellent performance.In addition to obtaining a lower detection limit,it takes only 30 minutes to complete the detection.Through further research,this method is expected to be applied to the rapid detection of Salmonella in actual samples.A novel method for the detection of Salmonella in microfluidic chip by glycosylated magnetic microspheres sorting-DEP separation-EIS in situ detection was established.The Glass-PDMS composite microfluidic chip contain inlet zone,“Tesla”structure mixing zone,detection zone and sampling zone were designed and prepared.In the experiment,a mannoside modified magnetic microsphere Phenyl-Man@Fe₃O₄ was prepared by reacting of an amino group-containing mannoside with a carboxyl group-containing ferroferric oxide magnetic microsphere,the average diameter of microsphere was 200nm.In the"Tesla"mixing zone,the synthetic bacterial sample was thoroughly mixed with the glycosylated magnetic microspheres,and the bacteria labeled by glycosylated magnetic microspheres were captured by dielectrophoretic?DEP?force in the detection zone containing the interdigitated array microelectrodes.Salmonella was enriched between the interdigitated electrodes,and the concentration of bacteria was detected by EIS.The results showed that the impedance change value?Z and the bacterial concentration[C]showed a good linear relationship in the range of 100⁵×10⁴CFU/mL,the detection limit was 100 CFU/mL.The detection limit was as reduced by one order of magnitude compared with the other DEP-EIS methods reported in the literature.It demonstrated that the glycosylated magnetic microspheres simultaneously have the function of EIS detection signal amplification.The established method shortens the detection time within 1 hour,and has great potential application value in the rapid detection of pathogenic bacteria and the rapid diagnosis of infectious diseases.