A Microbial Fuel Cell Based Biosensor For Cu²⁺ Detection

China's economy has developed rapidly in the past 30 years and has gradually realized the modernization of agriculture and industry.However,agricultural and industrial processes produce a large amount of waste water,which causes serious heavy metal pollution in soil,lakes and even offshore areas,and further affects environmental safety and balance.Among them,copper pollution is one of the most common heavy metal pollution.Due to its large pollution degree and wide pollution scope,it is easy to accumulate in fish intestines,plant roots and human body tissues through the accumulation of food chain in the ecological network,thus causing immeasurable influence on the balance and stability of the natural environment.The traditional copper ion detection method is based on Copper reagent method,atomic absorption spectrometry and bioluminescence method,etc.,which has a series of disadvantages,such as cumbersome operation,high price,the need for specific instrument detection and the inability for real time monitoring of copper pollution.In recent years,Microbial fuel cell?MFC?has been focused and studied by many researchers.Electrochemical microbial is innocubated in the anode chamber as a catalyst for organic matter,and than the chemical energy in anode chamber will be transfed into electricity by biological function.Electron acceptor was introduced in cathode chamber at the same time.Through the external circuit,elctrons will transfer to the cathode chamber.The protons generated by pyruvic acid circulation and the TCA cycle on the biochemical process will also enter into the cathode chambe by proton exchange membrane?or cation exchange membrane?,which finally mediates the separation of positive and negative charge.Based on the biochemical effect of electrochemical microorganisms in MFC,chemical signals in water can be easily converted into electrical signals,which can not only provide power output while wastewater treatment,but also provide a new idea for the construction of a biosensor based on environmental factors.Under the action of MFC,chemical signals in the environment are reflected as electrical signals,which can be further connected to the water monitoring system through the power network,providing a real-time signal transmission device for the construction of the water pollution warning,monitoring and processing system.In addition,the conversion between chemical signals,electrical signals and digital signals allows MFC sensors to be further integrated into the information network.Many studies have shown that the content of riboflavin synthesized by anodic electrochemical microorganisms is positively correlated with the electrical signal level of MFC output.Meanwhile,it has been confirmed in Escherich coli?E.coli?that ribB gene is the rate-limiting gene for bacterial riboflavin synthesis.Overexpression of ribB gene can relieve the negative feedback regulation of E.coli and improve the synthesis level of riboflavin.However,the extracellular membrane?Outer membrane,OM?of E.coli is a transport barrier with low permeability,which limits the shuttling of riboflavin as electron shuttles between cell membranes,thus reducing the efficiency of Excellular electron transfer?EET?of MFC.By introducing the porin synthesis gene OprF of P.aeruginosa PAO1 into E.coli,the membrane permeability level of E.coli can be enhanced by providing water channel protein.Finally,a molecular biosensor for Cu²⁺detection based on MFC specificity was designed and applied.In E.coli's response system to environmental factors,the two-component response system?TCS?is often used to perceive the changes of the surrounding environment and control the expression of related genes to respond to the changes of signal molecules.CusC promoter?PcusC?with specific response to Cu²⁺was selected from the genetic circuit of the E.coli two-component system CusSR,and it was fused to the upstream of T7 lysY gene to make the expression levels of these two gene respond to Cu²⁺induction in water,so as to change the electrical signal level of MFC by changing the synthesis and transport levels of riboflavin.The experimental results show that there is a linear relationship between the maximum voltage of the MFC biosensor and the Cu²⁺concentration between 0 and 0.7 mM.In addition,the operating parameters of the MFC sensor were tested and optimized to detect the interference degree of other heavy metals in the water.Finally,the application of Cu²⁺biosensor based on MFC was studied.At the same time,a portable water Cu²⁺pollution monitoring system is developed to provide real-time data support for water pollution warning,remote monitoring and treatment.