Construction And Application Of Whole-Cell Biosensor For Mercury Ion Detection

With the rapid development of modern industry,more and more mercury ions are discharged along with industrial waste and invade into the living environment of human beings.As toxic heavy metal ions,even the presence of trace amounts of mercury ions can cause significant biological toxicity to the human body,and severe cases can lead to death.Based on the needs of human life and health protection,a variety of mercury ion detection methods and devices have been continuously researched and developed,but most of the detection methods have poor selectivity to mercury ions,low sensitivity,and the detection process requires the help of large instruments,and the analysis time is relatively long.Defects such as long and complicated operation are not conducive to the efficient and convenient detection and rapid analysis of trace mercury ions,and there are great limitations in practical applications.Therefore,it is of great significance to develop high-efficiency,sensitive,low-cost,and easy-to-operate mercury ion specific detection technology.The whole cell biosensor integrates signal sensing elements and output elements.It uses microbial cells as biological materials and uses its own biosynthetic function to specifically identify the analyte and generate corresponding response signals.It has stable performance and strong environmental adaptability,Convenient operation and other advantages,but still need to be improved and improved in terms of sensitivity,selectivity and applicability.Based on these needs,this study uses the metal regulatory protein Mer R response regulation mechanism and the fluorescent protein mutants modified by protein engineering technology to construct a variety of excellent mercury ion whole-cell biosensors,and apply them to the actual water environment system with low content or even low content.Efficient detection of trace mercury contamination.The main research content and work are as follows:(1)With the visual detection of Hg²⁺in contaminated water as the research goal,genetic engineering methods are used to regulate the expression of downstream pyocyanin synthesis genes in Pseudomonas aeruginosa through the"transcriptional activation"mechanism of the metal regulatory protein Mer R to synthesize Pyocyanin is used as the detection signal of the colorimetric biosensor to achieve specific and visual detection of Hg²⁺.The colorimetric biosensor has a good linear relationship in the range of 25-1000 nmol/L,and the detection limit is 10 nmol/L.At the same time,the colorimetric biosensor has good metal selectivity and can resist the interference of metal external ions,and still has good performance in a wide p H range(p H 4-10).(2)The above-mentioned whole-cell biosensor for detecting Hg²⁺by colorimetric method requires pathogenic Pseudomonas aeruginosa to express the synthetic gene of pyocyanin,and the sensitivity needs to be improved.Therefore,based on the transcriptional activation mechanism of the metal regulatory protein Mer R and the microbial cell surface display technology,this chapter designs a mercury ion biosensor for Hg²⁺detection with the red fluorescent protein m Cherry as the reporting module to improve the problems of the colorimetric biosensor.The detection system of the sensor can quickly realize the visual detection of Hg²⁺in water samples,and has excellent selectivity and sensitivity to Hg²⁺,and its detection limit is as low as 0.1nmol/L.By fully simulating and utilizing microorganisms,the engineered biochemical system has great application potential in the detection of Hg²⁺in contaminated water.(3)In order to develop a more sensitive mercury ion biosensor,based on the work of the previous chapter,this chapter adopts a dual signal enhancement strategy,using a fluorescent-enhanced GFP mutant that responds to Hg²⁺as the reporter module to construct a signal-amplified mercury ion biosensor.Realize the detection of low concentration or even trace mercury in the water environment.The biosensor shows a response to Hg²⁺in a relatively wide range of 1-10000 nmol/L.Compared with most biosensors with similar structures,its detection limit is improved by one to two orders of magnitude.At the same time,the biosensor has good metal selectivity and still has a good response to Hg²⁺in a wide p H range(p H 4.0-10.0).The signal amplification strategy performed by the report module of the biosensor will be widely applicable to many other biosensors,providing an effective way to improve the performance of the biosensor.(4)In order to improve the response speed of mercury ion signals,this chapter aims to develop a whole-cell biosensor that uses a single protein as both a signal sensing element and an output element.By modifying the environment of the fluorescent protein m Cherry chromophore,the fluorescent protein is modified to be mercury-resistant.The ion-sensitive sensor is displayed on the surface of Escherichia coli through microbial cell surface display technology,and it is applied to the rapid visual detection of mercury pollution.The biosensor has a good response to Hg²⁺at the micromolar level,has good metal selectivity,and at the same time still has a good response to Hg²⁺in a wide p H range.In addition,the use of microbial immobilization technology to prepare cell-sodium alginate hydrogel test paper to achieve on-site visual detection of mercury pollution.The cell-sodium alginate hydrogel test paper can detect mercury pollution within 5 min,the operation process is simple,the equipment is cheap,and the test paper can stably maintain the fluorescence activity for 24 h at 4°C,which can be used as a high-throughput screening tool Initially reported the presence of mercury pollution in the natural environment.