| With the explosive growth of the world’s population and the rapid development of urbanization and industrialization,the human activity induced toxic chemicals release has been greatly increased.As the gatekeeper of ecological environment,environmental analysis is also facing greater challenges due to the increasing types of environmental pollutants,requirements for trace detection,and the complexity of the morphology and matrix of the analyte.Traditional environmental analysis focus on heavy metal ions,inorganic non-metal compounds(sulfide,nitrogen oxide,etc.),organic pollutants(pesticide residues,antibiotic release,etc.)and pathogenic microorganisms.With the development of human industry,the types of harmful substances are also increasing.The hot spots in environmental analysis can be divided into two categories:1.Environmental safety:Previous environmental issues mainly concentrated on natural factors such as geology,meteorology,hydrology,and soil biology.In addition to traditional pollution sources,novel industrially synthesized chemicals often also maintain environmental toxicological effects.With the help of bioaccumulation effect,negative impact would eventually appear on human society,which leads to a huge obstacle to environmental safety.Due to industry safety accidents or the release of pollutants,hazardous materials enter water,soil and atmosphere leading to a sudden disaster to environmental quality,endangering human society and the ecological environment,and causing environmental safety incidents.For example,the detection of explosives is very important in homeland security and environmental safety.Besides,the detection of trace antibiotics releases in the natural water and living organisms,and the detection of organic pesticide residue in the ecosystem,are all the cases of utilizing environmental analysis strategies to maintain environmental safety.Trace concentration,matrix interference and complex migration and transformation provide critical quest for the detection of environmental pollutants and related compounds;2.Biology Security:With the advancement of science and technology,modern biotechnology(genetic engineering and genome editing)has a remarkable impact on the ecosystem.China formally implemented the Biosecurity Law of People’s Republic of China,on April 15,2021.The first chapter clearly mentions that one of the purposes of the biosafety law is to "promote the construction of a community with a shared future for mankind and realize the harmonious coexistence of human and nature",which is suitable for protecting biodiversity,coping with microbial resistance,etc.In fact,from genetic engineering to genome editing,more and more biotechnologies are applied in environmental-related fields such as the optimization of plant traits and targeted mutagenesis of microorganisms.There are 189.8 million hectares of land planted with genetically modified crops in whole world.The environmental risk assessment(ERA)has become a nonnegligible issue that must be studied in genetic engineering of plants worldwide.And gene editing technology is also widely used in genetic modification of plants and microorganisms.Gene editing technology helps plants resist drought and disease more effectively,increases crop yields,and increases the absorption of air pollutants.Besides,it also applied in microorganism traits regulation,microbial fermentation technology and biofuels production.It is not difficult to find that the biosafety issues accompanying modern biotechnology will also affect the sustainable development of the ecological environment.The monitoring of biosafety is also a necessary topic that cannot be ignored in environmental analysis.As a powerful element detection method,inductively coupled plasma mass spectrometry(ICP-MS)detection has the advantages of high sensitivity,low matrix effect and high spectral resolution.With metal stable isotopes as exogenous labels,ICPMS detection and biological recognition based bioassay has been widely used in the detection of proteins,nucleic acids,biological enzymes,and small biological molecules,presented excellent analytical performance in clinical diagnosis and molecular biology.From element chelated tags to metal nanoparticles and polymer tags,ICP-MS detection based biosensing has continuously broken through in ultrasensitive bioassay.Thanks to the excellent spectral resolution,multiplex tagging strategies become accurate and reliable.In addition,the low matrix effect of ICP-MS detection can also overcome the complexity of the actual environmental samples.Plus,high designability from molecular biological recognition structures such as aptamers and antigen-antibody specific binding can used to construct various biosensing with high specificity and anti-interference capability.Up to now,there have been few reports about ICPMS detection and biological recognition based environmental analysis.Also,the revolution and evolution in methodology is still required.For example,ICP-MS can provide absolute quantitative results in lab,while environmental analysis often requires in field analysis.Traditional biological recognition based on enzyme,DNA,immunoassay,and whole cell recognition structure.Novel sensing platforms(such as gene editing sensing)can provide brand new biological recognition platform and further improve the analytical performance.This thesis is dedicated to establishing a novel CPMS detection and biological recognition based environmental analysis strategies,including DNA templated copper nanoparticle based "label-free" assay and gene editing biorecognition based environmental analysis with element reporter.Besides,we also aim to optimize analysis performance,improve sensitivity,shorten analysis time.Most importantly,we apply these two analytical platforms to the trace explosive residues detection in natural water,the bioaccumulation effect of antibiotics in fish,and the gene editing evaluation.The specific research content is as follows:1."Fluor/ICP-MS" bimodal trinitrotoluene detection based on DNA templated copper nanoparticles.Trinitrotoluene(TNT)is a world widely used explosive.The detection of TNT in soil and water environment is not only of homeland security value,but also an important issue of environmental security.Based on the label-free synthesis of DNA templated copper nanoparticles(ssDNA-CuNPs)and recognition of nucleic acid aptamers,we have established a label-free TNT biosensing strategy with both in field fluorescence detection and laboratory ICP-MS quantification.In the fluorescence mode,with rapid label-free synthesis of ssDNA-CuNPs,the fluorescence sensing unit synthesis and signal acquisition can be completed within 180s.The operation is facile and suitable for in field detection.As for the ICP-MS mode,it achieves a sensitivity of one part per trillion(PPT)with long time signal stablity,which is suitable for highly sensitive detection in the laboratory.The introduction of TNT nucleic acid aptamers ensures excellent selectivity and avoid interference by other explosives with similar physical and chemical properties in both modes.By adjusting the sequence of aptamers,we can apply this ssDNA-CuNPs based bimodal biosensing system to more explosives and environmental pollutants as a universal platform.2.Metal isotope detection and CRISPR/Cas14 biorecognition based aqueous antibiotic detection.As a revolutionary achievement of genetic engineering,the gene editing system is also an emerging biorecognition platform,which is expected to apply in environmental analysis.We propose a metal isotope detection and CRISPR/Cas14 biorecognition based aqueous antibiotic detection.To realize sensitive aqueous antibiotic detection,we chose CRISPR/Cas14 as the target sensing platform with the collateral cleavage ability of ambient ssDNA probe.We have purified and characterized the synthesis of element reporter,Tb-Rep.After optimizing the cleavage activity of metal tagging ssDNA probe,the element reporter based CRISPR/Cas14 biosensing can further improve the sensitivity with a better multiplex potential compared with the traditional "fluorescence-quenching" probe-based detection.After optimizing the various parameters,this method can obtain a detection limit as low as 2.06 nM in the detection of ampicillin and has excellent anti-interference performance.The real sample detection results also shown a good performance in the detection of complex matrices.This is also the first report of gene editing biosensing platform used with element reporter and ICP-MS detection.3.A fast and ultrasensitive antibiotic detection platform based on element reporter and CRISPR/Cas12a biorecognition:application in kanamycin bioaccumulation effect research.The previous CRISPR/Cas14 recognition based bioassay has the shortcomings of relatively low turnover,large amount consuming and time-consuming,which is still not satisfying for practical applications.We further proposed a fast and more sensitive element reporter based CRISPR/Cas12a biorecognition strategy and applied it to the detection of the geographic distribution and bioaccumulation effects of antibiotics in Pseudorasbora parva.Compared with the preliminary exploration of Cas14,the detection time is shortened to 30 minutes based on the CRISPR/Cas12a biosensing system.And the detection limit is lowered from the nM level to 4.06 pM,also with the anti-interference performance.We have also applied this method to the detection of kanamycin in Pseudorasbora parva from different sampling sites in Jialing River.In addition,we also studied the feeding of kanamycin to Pseudorasbora parva under laboratory conditions.The research of bioaccumulation effect of different tissues found that the cumulative effect of kanamycin exhibited liver>muscle>plasma,which is also consistent with the metabolic pathway of kanamycin in the organism.4.Label-free CRISPR/Cas9 assay based on DNA templated copper nanoparticles enable site-specific detection.Gene editing technology helps plants resist drought and disease more effectively,increases crop yields,and increases the absorption of air pollutants.The large-scale application of such plants and microorganisms is very likely to cause irreversible problems to the environment,leading to harmful effects biological safety.We further extended the proposed ICP-MS detection based "label-free" biosensing to the cuttingedge gene editing enzyme.We proposed a label-free CRIPR/Cas9 assay based on DNA templated CuNPs(dsDNA-CuNPs)enable site-specific detection.As a labelfree detection,canceling label synthesis process also contributes to keep the cleavage activity of Cas9 enzyme,which makes it more suitable for accurate and highthroughput in vitro assay.Due to the highly sensitive detection capability of ICP-MS and the amplification effect of copper nanoparticles,the proposed strategy can achieve the detection limit of nanomolar level,solving the detection sensitivity bottleneck of Cas9 as a single-turnover enzyme.We successfully detected the effects of single-base mismatch of complementary strand,non-complementary strand and sgRNA by analyzing the cleavage activity of CRISPR/Cas9.By pre-screen the different gene sequences,the dsDNA-CuNPs based label-free CRIPR/Cas9 assay can be applied to the detection of off-target effect in-vitro. |
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