| The continuous advancement of human globalization and the rapid development of modern society have caused increasingly serious pollution of marine environment.The frequent occurrence of harmful algal blooms results to the death of fish and other marine organisms due to hypoxia.At the same time,HABs release a large number of harmful gases and biological toxins,which can poison or even kill human beings.This phenomenon not only seriously damaged the ecosystem of lakes,rivers and oceans,but also harmed the living environment of human beings.In the process of ecological environment management and protection,it is necessary to carry out daily monitoring of various important substances by rapid and easy technical methods.In this dissertation,to address the shortcomings in current quantitative detection methods of HABs andtheir biotoxins,using the discipline advantages of Electronic Science and Technology,combining new nano materials with chips,biosensors were constructed to achieve the rapid sensitive detection of HABs and their biotoxins.There are fluorescence energy transductions before and after fluorescence-labeled probes capturing the specific genes of HABs in the graphene oxide nanosheets system.The biosensor achieved highly sensitive quantitative detection of HABs based on fluorescent signals with detection limit 0.1 fM in one hour.Additionally,it achieved the simultaneous detection of multiple samples and types of HABs on the double-layer micro-nano biochips.Using the affinity change of Aptamer to AuNPs and biotoxin,the biosensor achieved rapid quantitative detection of marine biotoxins produced by HABs.The detection can be quickly obtained only through color changes obtained in 40 minutes,with the linear range between 10 fM and 0.1 μM,and the detection limit can reach 10 fM.The main research work and innovations of this dissertation include as follows:1.Combining new two-dimensional nanomaterials with traditional methods of molecular biology,this work designed and constructed high-performance biosensors,which is applied to the quantitative detection of common species and substances in marine biology.It provides new type of detection methods for marine biology and promotes the cross-fusion of Marine and other disciplines.2.Designed and constructed a GO nanosheets-based high-sensitivity HABs quantitative detection biosensor.The nucleic acid concentrations of HABs were converted into fluorescent signal,and the intensity of fluorescent signal depended on the concentration of HABs.The evaluation approach of the biosensor was developed based on the Stern-Volmer equation.Through the optimization of the detection conditions,the quantitative detection of HABs can be completed within one hour.The linear range of the 18S rDNA gene detection of Heterosigma akashiwo is from 0.1 fM to 100 nM,and the detection limit can reach 0.1 fM.The biosensor can identify one base mismatch in target gene,which achieves high sensitivity,high specificity and rapid detection of HABs.This method has advantages of a simple processing process,few experimental consumables,and low cost.3.Combining nano-sensing materials with micro-nano processing biochips,a double-layer micro-nano biosensor was developed,which can be used for the simultaneous detection of multiple samples and HABs.This double-layer micro-nano biosensor chips can detect at least six kind of HABs within one hour.Comparison with traditional molecular biology methods,the developed biosensor reduces the amount of detection reagents,the analysis cost,and the consumption of precious biological reagents.Additionally,the design of closed detection system can also reduces external pollution and operating errors,which improves the detection reliability.4.Designed and constructed an AuNPs-based highly sensitive quantitative colorimetric biosensor for detection of marine biotoxins producted by HABs.The biosensor took marine biotoxins as the target and converted the concentration signal of marine biotoxins by changing the aggregation state of AuNPs into optical signal.Through exploration and optimization of experimental conditions,the quantitative detection of marine biotoxins can be completed within 40 minutes with the detection linear range from 10 fM to 0.1 μM by using the developed biosensor,and the detection limit of this biosensor can reach 10 fM.Comparison with traditional detection methods,it has the advantages of simple operation,low cost,no need for cumbersome sample preparation,and rapid detection.It has broad prospects in the monitoring of seawater environmental pollution and seafood safety assessment. |
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