Untargeted Screening Technology For Gram-negative Bacteria Based On Biosensors

Food safety is a global issue at present,mainly manifested as constant occurrence of food contamination incidents.With the rapid development of food processing technology,the risks of food safety cannot be ignored.Therefore,the resulting foodborne diseases have become an important factor affecting public health.About 2/3 of foodborne diseases are reported to be caused by bacteria.Traditional methods for detection of bacteria include: culture and colony counting method,methods based on immunology and PCR methods.These methods are dependent on the specificity of microbial and biochemical identification,and they only be used to detect the known bacteria.There is a wide variety of bacteria,the limitations of the traditional detection methods lead to a higher missing detection rate of unknown bacteria,and the reliability of the test results is questioned to some extent.Therefore,it is urgent to establish a rapid and accurate method for the detection of unknown bacteria in food,and technique for rapid screening and toxicity evaluation of unknown bacteria.And biosensor technology has high sensitivity and strong specificity,in particular,cell sensing technology uses live cells as recognition elements,and can simulate the real reflection of the body,which has been widely used in medical diagnosis,drug screening,environmental monitoring and other fields.In this study,Gram-negative bacteria were studied,LPS signal transduction mechanism and AHL molecular imprinting recognition were used as theory evidence;electrochemical detection,preparation of recombinant protein,magnetic molecular imprinting and fluorescent reporter gene transfection were combined to build a series of new sensitive untargeted screening technology for Gram-negative bacteria,which had provided a new way for food safety detection and evaluation.In order to explore the process of electrochemical cell sensing to detect the secretion of NO of activated RAW264.7 cells by LPS of Gram-negative bacteria.Electrodeposition was used to modify the platinum nanoparticles on gold electrode surface,as well as innovative combination of new polymer carbon nano material graphene oxide,for preparation of NaAlg/GO gel with good biocompatibility and mechanical strength,RAW264.7 cells were fixed on the modified electrode surface to form cell 3D culture system.RAW264.7 cells were used as identification elements to construct electrochemical cell-based biosensor,and through the highly sensitive detection of LPS,indirect detection of Gram-negative bacteria was achieved.When the sensor was in the range of 10-2 ng/m L to 1 ng/m L,there was a good linear relationship between LPS concentration and peak current value,the linear equation was y=9.035x+53.578,R2=0.9842;when in the range of 1 ng/mL to 10 ng/mL,there was a good linear relationship between LPS concentration and peak current value,the linear equation was y=0.4778x+53.756,R2=0.9941;when in the range of 10 ng/m L to 104 ng/mL,there was a good linear relationship between LPS concentration and peak current value,and the linear equation was y=10.019x+50.3,R2=0.9732.The limit of detection of LPS was 8 pg/m L.The feasibility of the 3D cell electrochemical sensor for detection of LPS was verified,which realized the indirect detection of Gram-negative bacteria.To meet the need for rapid,sensitive and active direct detection of Gram-negative bacteria.The large number of natural high affinity IgE receptors(Fc?RI)on the surface of RBL-2H3 cells were used to combine with virulence factor receptor CD14-Fc? chimeric proteins synthesized by recombinant protein technology for the innovation application of fluorescence detection technology into detection of Ca2+ signal transduction mediated by IgE in RBL-2H3 cells stimulated by Gram-negative bacteria or LPS through gene encoding calcium indicator GCaMPs and biological ion sensing in series.When the sensor was in the concentration range of 101 CFU/mL~103 CFU/mL,with the increase of E.coli ATCC 25922 concentration,the relative fluorescence intensity of cell-based biosensor was increasing,the linear equation was y=1.4081x+0.0332,R2=0.9728.The limit of detection of E.coli ATCC 25922 was 102 CFU/m L.Pork was selected as food sample for the detection of Gram-negative bacteria in real samples.Compared with blank control group,the relative fluorescence intensity of 102 CFU/mL E.coli ATCC 25922,S.typhimurium ATCC 14028,and S.dysenteriae ATCC 3313 stimulated by fluorescence RBL-2H3 cells was significantly increased at a concentration of 102 CFU/mL.These results demonstrated the feasibility of using this cell-based biosensor for the detection of Gram-negative bacteria,and the detection time was short.In order to further realize the detection of Gram-negative putrefying bacteria,Fe3O4 beads were synthesized by solvothermal method,and quorum sensing signal molecular analog of Gram-negative bacteria was innovatively used as template molecule,taking Fe3O4 MNPs as kernel,SiO2 was shell,grafting NH2 was made on the obtained core shell type magnetic sphere surface,and its polymerization on the surface of the aminated core-shell magnetic spheres was made,for the preparation of multifunctional polymer microspheres with core-shell structure Fe3O4@SiO2-MIP.By magnetic adsorption,immobilization of magnetic molecularly imprinted polymer on the surface of magnetic electrode was achieved,gaining the magnetic molecularly imprinted electrochemical sensor for detecting signal molecules of Gram-negative bacteria.Fe3O4@SiO2-MIP was adsorbed by magnetic electrode and electrochemical analysis was performed,the DMHF concentration had a good linear relationship with the current in the range of 2.5×10-9~1.0×10-7 mol/L,the linear equation was y=-0.3095x+90.967,R2=0.9915,the detection limit was 8×10-10 mol/L.Seven kinds of bacterial supernatant were studied,AHL was not detected in the supernatant of E.coli O157:H7,S.typhimurium ATCC 14028 or S.aureus ATCC 29213,this result was consistent with the characteristics of these bacteria that did not produce AHL.Therefore,the feasibility and specificity of the method for the determination of AHL were illustrated.The quorum sensing molecules were detected in the sterile supernatant of A.hydrophila and P.aeruginosa,the recoveries were between 96.1% and 103.4%,which met the testing requirements.RSD value was less than 2.72%,within the permissible error range,which explained the method could be used to determine the reliability of AHL data.And the feasibility of the application of the magnetic molecularly imprinted electrochemical sensor for indirect detection of Gram-negative putrefying bacteria was verified.However,it was not enough to complete the detection of unknown bacteria,identifying the virulence was the ultimate goal.Fluorescent protein gene(mCherry)was linked after the NF-?B identification element for the construction of fusion plasmid.By liposome transfection,pGL4.26-mcherry-NF-?B plasmid were introduced into 293/hTLR4A-MD2-CD14 cells,for the construction of 293/hTLR4A-MD2-CD14 cell-based biosensor expressing red fluorescence.Hot phenol water method was used for extracting LPS of 14 Gram-negative bacteria,for SDSPAGE silver staining analysis,and it was used to stimulate the sensitive cell model,and the fluorescence and inflammatory factor secretion were compared and analyzed.20 h was used as end time of stimulus,with the increase of LPS concentration,the fluorescence intensity of E.coli,S.typhimurium,E.sakazakii,S.dysenteriae and P.aeruginosa LPS stimulation group increased gradually,the secretion of TNF-? and IL-8 also increased gradually.Under the same concentration of LPS stimulation,fluorescence intensity of LPS in P.aeruginosa(Pseudomonadaceae)was significant lower than that of other bacteria(Enterobacteriaceae)LPS stimulation groups,this was related to the five-acylated lipid A in P.aeruginosa.Therefore,the feasibility of evaluating the virulence of Gram-negative bacteria LPS by this cell-based biosensor was verified.Intestinal epithelial cells were the earliest sensors for bacterial invasion,therefore,it was very meaningful to study the response of intestinal cells to LPS.Western blot,flow cytometry,RT-PCR were used as technical means to detect the expression of protein and the expression of inflammatory factors,firstly,the expression of Caco-2,HT29,SW480 TLR4 in three kinds of human intestinal epithelial cells was studied,as well as intestinal epithelial cells response to LPS.Subsequently,intestinal epithelial cells Caco-2,HT29,SW480 were respectively cocultured with macrophage THP-1,and the dialogue between intestinal epithelial cells and laminae propria M? was imitated,the innovative study on expression of TLR4 in intestinal epithelial cells and intestinal epithelial cells response to LPS was conducted under the stimulation of LPS co-culture system.The results showed,under the condition of single culture,Caco-2 cells did not express TLR4,and intracellular expression of TLR4 in HT29 cells,SW480 cells had surface expression of TLR4.After LPS stimulation for 24 h,the expression of TLR4 in Caco-2,HT29,SW480 cells were not increased.Caco-2 and HT29 cells had no response to LPS,SW480 cells responded to LPS.Under co-culture system of LPS stimulation for 24 h,there were no significant increase in TLR4 expression in HT29 and SW480 cells,while the TLR4 expression of Caco-2 cells increased significantly.HT29 cells did not respond to LPS,and SW480 and Caco-2 cells responded to LPS.Therefore,SW480 cell was a sensitive cell model of LPS stimulation.The mechanism of LPS stimulation of intestinal cells was explored,which provided theoretical basis for screening LPS sensitive cell model,and new idea for the next step to construct the co-culture model.To sum up,in this paper,we focused on the application of biosensor technology in untargeted screening for Gram-negative bacteria.Gram-negative bacteria were detected,combined with electrochemical detection,recombinant protein preparation,fluorescent reporter gene transfection and magnetic molecular imprinting technology,a series of new,active and real biological sensor detection and evaluation methods were constructed to be applied to the actual sample detection,which provided a strong guarantee and support for the improvement of the recognition level and the development of food safety detection and evaluation technology.