Bacteria-Immobilized Preparation As The Electrochemical Biosensor For Antibiotics Susceptibility Test

ObjectiveWith the continuous development of medicine and health care,drug quality and safety testing attract more and more people's attention.There are some disadvantages of traditional antibiotic drugs tests,such as complicated operation,high cost and low selectivity,which all induce limited application.In order to remedy deficiencies of the traditional antibiotic drugs tests,it is important to develop a low energy-consumption,simple and fast method.The immobilized microorganism,with some special advantages,such as good stability,easy conservation,convenient operation and low cost,which has been widely applied in the fields of the drug quality control and water quality rapid detection.In this study,we attempted to oxidize graphite rod to harvest graphene oxide?GO?on the surface of graphite,and then Escherichia coli?E.coli?was fixed on the GO surface.Polyvinyl alcohol?PVA?and commercially available GO sample were mixed to prepare colloid mixture for embedding E.coli fixed on the GO surface.In addition,sodium alginate?SA?and MIL-53?Fe?are doped into polyvinyl alcohol?PVA?to form a complex colloid,which was cross-linked with boric acid to obtain MIL-53?Fe?/E.coli/SA/PVA particles.To combine the electrochemical techniques,treated E.coli should be used to construct a new method of antibiotic drugs test in a sensitive and rapid way.MethodsAs used herein,a quality control strain of E.coli was used as a microbial target,and K₃[Fe?CN?₆]was a probe to transport electrons of microbial respiration generated product to the electrode surface.In order to measure the efficiencies of the drugs,the changes of microbial activities had to be gotten by monitoring the quantity changes of reduced product([Fe?CN?₆]^4-).In this paper,some optimal conditions,such as the fixed time of target microorganism,the concentration of GO in the colloid mixture,the stored method and the stored time of immobilized microorganism,were studied seriously.All optimal conditions were used to immobilize the E.coli cells that were applied to test the efficiencies of different antibiotic drugs.In this study,the sensitive bio-probes were prepared by optimizing the formation of GO/PVA/E.coli/GO/GR particles and MIL-53?Fe?/E.coli/SA/PVA particles in different conditions.The immobilized microbial sensor was fabricated and applied to the detection of different antibiotics.ResultsGO/PVA/E.coli/GO/GR particles:the optimal immobilized time was screened in 16h.For screening the embedding materials,the optimal content of GO was 0.5mg/L when selected content of PVA was fixed in 10mg/L.The optimal crosslinking time was 1min by studying the boric acid effects on the bioactivities of immoblilized E.coli.Comparing the changes of bioactivities of immobilized E.coli depending on the stored time under the different stored conditions,4?refrigerator dry stored condition was more suitable for PVA-GO composites immobilized E.coli.In the drug susceptibility testing,the concentration of 50%inhibition of tested antibiotic drugs that were gentamicin,amikacin,amoxicillin,cefepime were 8.01mg/L,6.51mg/L,9.93mg/L and4.07mg/L,respectively.For the drug susceptibility testing of vancomycin,the highest inhibiting rate were 32%at its tested concentration of 16mg/L.MIL-53?Fe?/E.coli/SA/PVA particles:the optimal concentration of colloid-doped MIL-53?Fe?was 0.05g/L.The optimal crosslinking time was determined to be 1h,and the samples were stored under dry conditions at 4°C.In the toxicity test,the IC50 of DCP against MIL-53?Fe?/E.coli/SA/PVA is 27.67mg/L.And in the antibiotic drug test,The concentration of the gentamicin,amikacin,and vancomycin whose inhibition rate reaches 50%is 14.12mg/L and 16.01mg/L.ConclusionIn this study,the two immobilized microbial particles were prepared by optimizing the formation in different conditions.The sensitive and reliable immobilized microbial sensor was fabricated and applied to the detection of standard toxicant DCP and different antibiotics.The results showed that the immobilized microbial particles doped with nanomaterials had high response in the detection of toxic DCP and antibiotics.Immobilized microorganisms could be stored for long-term storage and used conveniently,and the electrochemical sensors based on immobilized microorganisms are more time-saving and convenient than traditional detection methods.In summary,the electrochemical microbial sensor is sensitive,convenient and time-saving,and has potential application in the field of antibiotic effectiveness detection.