Application Of Electrochemical Microbial Sensor In The Field Of Toxicity And Drug Susceptibility Experiments

Objective In recent years,with the continuous progress of science and technology,it not only brings changes to our life,but also brings a huge burden to our environment.Water pollution is particularly serious,while toxic metals and medical drugs are two important factors causing water pollution that cannot be ignored.Therefore,it is of great significance to find a conventional method to monitor and control pollutants.The traditional water quality monitoring operation is complex,time-consuming and demanding.Taking higher animals as receptors increases the uncertainty of experimental results,and the cost of time,human and material resources is very high.In this study,a simple electrochemical method was proposed to determine the content of bacteriostatic substances using microorganisms as receptors.In this study,the glassy carbon electrode was modified by dropping coating with neutral red(NR)doped nanomaterials,and the immobilized media test system was established.Using E.coli as the test strain,electrochemical biosensor was prepared by immobilization and non-immobilization methods,and introduced into common metal ions,such as Zn2+,Cu2+,and standard toxicity test of 3,5-dichlorophenol(DCP),as well as antibiotic sensitivity test of cefepime and streptomycin.It is hoped that through the continuous optimization and improvement of the sensor,a microbial electrochemical sensor with rapid response and high sensitivity to environmental stress caused by chemicals and antibiotics can be obtained.It can achieve simple preliminary detection and follow-up microbial recovery and treatment with low technical cost and meet the requirements of low-carbon technology development with low energy consumption,low emissions and low pollution in China.Methods In this study,NR as the medium and graphene(GR)as the conductive material were doped in the swelling film forming material polyvinyl alcohol(PVA)to form a composite film droplets coated on the surface of glassy carbon electrode(GCE)to prepare a biosensitive probe for bacterial activity detection.The modified electrode PNR/GR/PVA/GCE was prepared by electropolymerization to form polyneutral red(PNR).The effects of three factors on the performance of PNR/GR/PVA/GCE composite electrode were investigated by orthogonal experiment and variance analysis.Cyclic voltammetry(CV)was used to optimize the polymerization conditions of modified electrode(electrolyte concentration,number of scanning segments),and an electrochemical method for in vitro microbial activity detection was established using suspended bacteria as a model.On this basis,the existence state of the microbial model was changed,and the microorganisms were doped in the film-forming material and fixed on the electrode surface to form a new biocomposite membrane PNR/GR/PVA/E.coli/GCE sensor probe.The orthogonal experiment was still used to screen the optimum ratio of the four factors.PNR/GR/PVA/E.coli/GCE under optimal conditions were applied to the subsequent determination of biological activity.Results(1)Segment 30,Na NO3 concentration of 0.5 mol/L is the best polymerization conditions of NR/GR/PVA composite membrane,the final concentration of NR is 4 mg/m L,the final concentration of GR is 4 mg/L,The final concentration of PVA at 6%(W/V%)was the best condition for the preparation of modified electrode NR/GR/PVA/GCE(2)When escherichia coli was suspended,the sensitivity to antibiotic detection was highest when OD600=1.(3)Combined with PNR/GR/PVA/GCE prepared under optimal conditions for the toxicity detection of antibiotics cefepime,streptomycin,vancomycin,heavy metal ions Zn2+,Cu2+and standard DCP.Cefepime and streptomycin reached 30% inhibition rate on E.coli at 4.85 mg/L and 13.46 mg/L,respectively,while vancomycin had poor inhibition effect.The IC50 of Zn2+,Cu2+and DCP to E.coli were 13 mg/L,4.5 mg/L and 7.5 mg/L,respectively.(4)The optimum conditions for the preparation of BIofilm electrode PNR/GR/PVA/E.coli/GCE were as follows: the absorbance of bacteria solution OD600=4,the final concentration of NR/GR/GR 4 mg/L and the final concentration of PVA 6%(W/V%).(5)The biofilm electrodes prepared under the optimal conditions were used for cefepime and streptomycin,and the IC50 of E.coli were 1.5 mg/L and15 mg/L respectively.The IC50 of vancomycin was poor,and the IC50 of Zn2+reached at 10 mg/L.Cu2+and DCP were 3 mg/L and 4 mg/L respectively,and the inhibition rate was more than 60%.(6)The combination of antibiotics cefepime and streptomycin had a synergistic effect,and the combined toxicity of heavy metal ions Zn2+and Cu2+was antagonistic.Conclusions The PVA/PNR/GR/GCE electrochemical sensor was prepared by using suspended bacteria as model under the best conditions.It has the advantages of high sensitivity,good repeatability,strong stability and repeatability.Compared with the traditional biological activity detection,it is more rapid and convenient to judge the toxicity of antibacterial substances by observing the size of the current.In addition,this method can truly reflect the biological toxicity of heavy metals in natural water,and is suitable for long-term monitoring and detection of environmental water quality.The PNR/GR/PVA/E.coli /GCE biosensor formed by bacteria fixed on the electrode is easier to operate than the suspended bacteria method and has higher sensitivity.It can detect trace toxins.The two methods complement each other to expand the detection range of concentration of bacteriostatic substances,so it has a good application prospect in water quality detection.