The Research On Detection Technology Of Heavy Metals On Paper-based Chip Based On Allosteric Transcription Factors

Objective At present,with the rapid development of industry and agriculture,the pollution of heavy metals in China is becoming increasingly serious.Different from organic compound pollution,heavy metal ions cannot be degraded in the environment.Aside from their non-biodegradability,they can impact the human body due to their biological concentration in the food chain.The allosteric transcription factors（a TFs）,which widely exist in prokaryotic organisms and are responsible for regulating the metabolism of small molecules,are used as novel recognition elements in the construction of biosensors.Cell-free biosensors based on a TFs have the advantages of simple preparation,rapid response and strong specificity.Therefore,it is of practical value to develop and design a new sensing technology to detect heavy metal ions in water with a TFs as the recognition element.In addition,as the carrier of cell-free biosensors,paper-based materials have the advantages of low cost,convenient storage and portability,and are convenient for on-site detection.In this study,mercury,lead,cadmium and arsenic,which are common toxic heavy metals in water,were used as the research objects.Based on the interaction between the a TFs and double-stranded DNA（ds DNA）,combined with the cell-free in vitro transcription（IVT）system,a cell-free fluorescent RNA sensing technology for heavy metal detection was constructed with the allosteric transcription factor as the recognition element,and the sensitivity was further improved by the rolling circle transcription（RCT）.In addition,a paper-based chip with multiple reaction cells was designed and developed to realize the simultaneous detection of multiple heavy metal ions under field conditions.The research is as follows.Methods 1.Research on cell-free sensing technology for detecting heavy metals based on a TFs.Combined with the cell-free transcription system expressing fluorescent RNA in vitro,a new sensing technology for heavy metal ions detection based on a TFs was designed.First,a cell-free in vitro transcription system was constructed using ds DNA containing T7 promoter,allosteric transcription factor binding site,3WJd B expression gene and T7 terminator as reaction template.Then,the a TFs were screened and characterized with surface plasmon resonance（SPR）and electrophoretic mobility shift assay（EMSA）.Next,the appropriate concentration of a TFs were added into the cell-free in vitro transcription system to build a cell-free biosensor.Furthermore,DNA logic circuit was designed to eliminate the signal crosstalk.Finally,the practicability of this method was verified by the detection of actual water samples.Methods 2.Research on the ultra-sensitive detection technology of heavy metals based on a TFs and using RCT as the fluorescence signal amplification method.An ultra-sensitive detection method for heavy metal ions in water based on a TFs was designed by using the signal amplification method of RCT.First,a single stranded circular DNA with local double strands was designed to meet the requirements of the binding of a TFs and the process of RCT reaction.Then,the feasibility of the detection principle of this method was characterized by EMSA and fluorescence measurement.Next,orthogonal experiments were carried out to optimize the content of a TFs in the detection system.Finally,DNA logic circuit was designed to eliminate the signal crosstalk.Methods 3.Research on the paper-based chip for rapid and multiple detection of heavy metals in water.The designed microfluidic chip equipped with freeze-dried paper-based biosensor was used to realize the simultaneous detection of four heavy metals under field conditions.First,the detection limit of the cell-free biosensor was adjusted by changing the amount of a TFs.Then the paper-based material and its pore size were screened.Next,the cell-free biosensor was equipped on the paper-based material by freeze-drying to construct the paper-based biosensors.Finally,the paper-based biosensors for detecting different heavy metal ions were placed on the designed microfluidic chip to build a paper-based chip.Results 1.Research on cell-free sensing technology for detecting heavy metals based on a TFs:Under the best conditions,the detection of 0.5 nmol/L Hg²⁺,0.1 nmol/L Pb²⁺,10 nmol/L Cd²⁺and 1 nmol/L As³⁺could be realized through the fluorescence results observed by naked eye.The Pb²⁺signal crosstalk in the Cd²⁺detection system was successfully solved by the introduction of DNA logic circuit.The recovery rate of heavy metal ions in river water was 91.09-123.24%,and the relative standard deviation（RSD）was not more than 6.71%,indicating the potential of this sensing technology in practical application.Results 2.Research on the ultra-sensitive detection technology of heavy metals based on a TFs and using RCT as the fluorescence signal amplification method:Under the optimal detection conditions,the fluorescence value generated by this detection system had a good linear relationship with the logarithm of the concentration of heavy metal ions.The linear equations were Y=217.5*X+534.6（R²=09918）for Hg²⁺,Y=161.5*X+617.7（R²=09856）for Pb²⁺,Y=183.8*X+463.6（R²=09908）for Cd²⁺,Y=177.3*X+549.3（R²=09821）for As³⁺,where Y was the fluorescence value,and X was the logarithm of the concentration of heavy metal ions,and the detection limits were 0.034,0.006,0.135 and 0.016 nmol/L（3σ/S）,respectively.The sensitivity was much higher than the previously constructed cell-free biosensor.Results 3.Research on the paper-based chip for rapid and multiple detection of heavy metals in water:The cellulose acetate membrane with a pore size of 0.2μm was selected as the paper-based material for the construction of paper-based biosensors.Next,the detection limits of Hg²⁺,Pb²⁺,Cd²⁺and As³⁺were adjusted to 5 nmol/L、48nmol/L、45 nmol/L and 128 nmol/L respectively,according to the domestic drinking water standard of China.Finally,the qualitative detection of four heavy metal ions was realized under the condition of on-site detection using the constructed paper-based chip.Conclusions In this study,a TFs were used as recognition elements,which were combined with cell-free in vitro transcription system and RCT reaction respectively to realize visual detection and ultra-sensitive detection of heavy metal ions in water.It made the generation of fluorescent signals faster and the construction of detection system simpler by using fluorescent RNA as signal output.In addition,a paper-based chip was constructed based on paper-based biosensors and microfluidic chip,which realized multiple detection of heavy metals in water under field conditions.The results of this study proved the feasibility of the two sensing technologies and the practicability of paper-based chips which provided a new means for monitoring heavy metals in environmental water and a new solution for high-throughput detection of other contaminants.