Microbial Biosensor Construction Based On Recombinant Proteins Expression For The Detection Of Catechol And Sulfide

Microbial biosensor,a novel test method,with several advantages such as good stability,simple preparation,long service life and low cost has become a research hotspots in the field of environmental detection.In the construction of microbial biosensor,living microorganism is used as bio-recognition element,which provide natural pH value,cofactors(electronic donors such as NADH,etc.)and energy for enzymatic reaction.Due to microorganism as bio-recognition element,the microbial biosensor will remain stable for quite a long time.Moreover,the biological components of microbial biosensor can be regenerated by immersing in the medium.Therefore,the biological activity of the biological components can be remain stable for a long time,and extended the service life of the microbial biosensor.However,there are several technological limitations such as the weakly catalytic ability of the identification element and lack of effectively immobilized carrier materials.Thus,how to solve these problems and take advantage of microbial biosensor for environmental pollutants detection is the research direction in the future.In this paper,the carE gene,which belongs to Sphingobium yanoikuyae XLDN2-5,was cloned into Escherichia coli BL21(E.coli BL21)cell and expressed catechol 2,3-dioxygenase.For the construction of a microbial biosensor,the recombinant E.coli BL21-C23O was selected as the bio-recognition element for catechol detection.Nanoporous gold(NPG)with its unique properties was selected for recombinant E.coli BL21 cells immobilization,and then a glassy carbon electrode(GCE)was modified by the resulting E.coli BL21-C23O/NPG biocomposites to construct an E.coli BL21-C23O/NPG/GCE bioelectrode.The sensitine detection of catechol was attibuted to the co-catalysis of E.coli BL21-C23O and NPG.Linear responses were obtained for catechol concentrations ranging from 1 to 150 ?M and 150 to 500 ?M with the sensitivity of 332.24 ?A·mM-1·cm-2 and 107.68 ?A·mM-1·cm-2,respectivily.This microbial biosensor also has a low detection limit with 0.24 ?M.Furthermore,the E.coli BL21-C23O/NPG/GCE bioelectrode also had a strong anti-interference ability.Beisdes,the resulting bioelectrode was successfully applied to the detection of catechol in the real sample.These unique properties make the E.coli BL21-C23O/NPG/GCE bioelectrode an application potential for the catechol detectionPromoting the specificity and electrocatalytic activity of sulfide is crucial in sulfide biosensor design.In this research,recombinate E.coli BL21-SQR cell,which has high catalytic activity to sulfide,was choosed as the bio-recognition element;NPG with high biocompatibility and catalytic ability to sulfide was selected as the immobilized cell carrier.The electrochemical reaction of sulfide on the surface of E.coli BL21-SQR/NPG/GCE bioelectrode was attibuted to the co-catalysis of E.coli BL21-SQR and NPG.Besides,the electron transfer process in the presence ofsulfide was a diffusion-controlled,irreversible process.Linear responses were observed for large sulfide concentrations ranging from 50 ?M to 5 mM,with a sensitivity of 18.35?A mM-1 cm-2 and a detection limit of 2.55 ?M.Furthermore,anti-interference test indicated that the resulting E.coli BL21-SQR/NPG/GCE bioelectrode presented strong anti-interference capability against some cations,anions,and organic pollutants,especially cyanide(CN-)which interfered significantly with sulfide detection using an enzyme-based inhibitive biosensor.For the detection of sulfide in wastewater,the data generated by E.coli BL21-SQR/NPG/GCE bioelectrode were in good agreement with those produced by the methylene blue spectrophotometric method.These unique properties make the resulting E.coli BL2 1-SQR/NPG/GCE bioelectrode an excellent choice for sulfide determination in practical application.For sulfide reliable detection,promoting selectivity and sensitivity is crucial in sulfide biosensor design.Here,a biosensor was constrcted by E.coli BL21-SQR/NPG/GCE bioelectrode and reduced Graphene Oxide(rGO).rGO with excellent conductivity and efficient electron traansport was selected to improve the sensitivity of this resulting sulfide micobial biosensor.The electrochemical oxidation signal generated by E.coli BL21-SQR/rGO/NPG/GCE bioelectrode was triple that of the E.coli BL21-SQR/NPG/GCE bioelectrode cunstructed in last chapter.Linear responses were observed for large sulfide concentrations ranging from 20 ?M to 5 mM,with a higher sensitivity of 23.5 ?A mM'1 cm-2 and a detection limit of 0.85 ?M.The load of rGO on the surfance of the microbial biosensor could dramatically improve the sensor property in sulfide detection.Further,the E.coli BL21-SQR/NPG/GCE bioelectrode presents strong stability,reproducibility,and anti-interference capability in detecting the the real seawater samples.These unique properties make rGO has great advantage in biosensor construction which an excellent choice for environmental pollutants determination in practical application.Based on above results,a microbial biosensor,which use the microbial cell overexpressing recombinant enzyme as the bio-recognition element,combines the advantages of both enzyme(efficient catalytic capacity;great sensitivity;excellent anti-interference)and microbial(good stability;simple preparation;long self-life;low cost)biosensor.Meanwhile,nanomaterial with unique properties of structure and function in the area of electrochemical sensor combine with these recombinational microorganisms to form microorganism/nanomaterial electrode.Such electrode combines the advantages of both abiotic electrochemical sensor,enzyme biosensor and microbial biosensor.A selective,sensitive and efficient biosensor for the detection of environmental pollutants will achieve by using the synergy of nanomaterial and recombinational microorganism.