Applications Of Fluorescent Biosensor Based On Molybdenum Disulfide In Nucleic Acid Analysis

Genetic information is stored in the form of the nucleotide sequence in nucleic acids,and the analysis of nucleic acid has important application in the field of clinical laboratory diagnosis,such as early prevention,early diagnosis of tumor,various related disease diagnosis,curative effect monitoring and individualized treatment,etc.Because specific sequences of nucleic acids can identify the target molecules or have some catalytic reaction,design and development of various fast simple nucleic acid analysis method based on the recognition of specific nucleic acid have long been a hot spot in the field of molecular diagnostics.Traditional nucleic acid analysis methods include capillary electrophoresis,denaturing high performance liquid chromatography,DNA microarray,denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis,etc.The methods are mostly based on PCR to achieve nucleic acid analysis.However,these methods have the following drawbacks:the need for more pure PCR products,the high experimental cost,the tedious experimental steps,time-consuming and so on.In recent years,some new nucleic acid analysis methods have overcome the shortcomings of traditional methods.Among them,DNA biosensors have been developed rapidly in the field of nucleic acid analysis because of their low experimental cost,high sensitivity and good specificity.Compared with other DNA biosensors,fluorescent DNA biosensors are widely used in the diagnosis of nucleic acid,because the sensor has its unique advantages,such as easy sample preparation,simple operation,rapid response,easy to use.Fluorescence probes play an important role in fluorescence sensing technology,in which aggregation-induced quenching（ACQ）is the biggest obstacle to the development of traditional fluorescence probes when applied to biosensing.In the late 1980 s and early 1990 s,nanotechnology was gradually applied to the field of biosensors.The application of nanomaterials is a branch of the most dynamic,promising and vaule of research in nano-biosensing technology.Since two British scientists Konstantin Novoselon and Andre K.Geim discovered graphene in 2004,graphene-liked two-dimensional nanomaterials,especially transition metal dichalcogenides（TMDs）,have been gradually paid attention in the application of biosensing.Compared to graphene,TMDs can be easily synthesized on a large scale and dispersed directly in aqueous solution without the need for additional complex treatments such as surfactants or oxidation.In addition,TMDs are potentially applied to the field of fluorescent nano-biosensor because of their excellent quenching ability.Molybdenum disulfide（MoS₂）is the most typical and commonly used representative material for TMDs.In this work,by combining the MoS₂ nano-sheet with fluorescent probe,one can effectively distinguish ss-DNA and ds-DNA owing to high quenching ability of MoS₂.After that,the fluorescent probe is improved,which is aimed at improving the sensitivity and specificity of detection.As such,a series of fluorescence biosensor technologies for simple rapid nucleic acid analysis including DNA methylation are developed.The specific research contents are as follows:（1）Rapid analysis of DNA methylation based on molybdenum disulfide fluorescence biosensorDNA methylation is an important tumor marker,and it is important to establish a simple,rapid and sensitive DNA methylation assay.BstUI is a restriction endonuclease that is sensitive to human 5’-CGCG-3’ sites,but its cleavage function is lost when this site occurs half/full methylation.Here,we present the development of a simple fluorescence biosensor based on water-soluble MoS₂ nanosheets and restriction endonuclease BstUI for methylation analysis of p16 promoter.The biosensing platform exhibited excellent sensitivity in detecting DNA with a linear range of 100 pM²0 nM and a detection limit of 140 pM.More importantly,the method could distinguish as low as 1% difference in methylation level.Compared with previous methylation analysis,the design is both time-saving and simple to operate,avoiding the limitations of PCR-based assays without compromising performance.（2）Preliminary study on DNA biosensors based on aggregation-induced luminescent probes and molybdenum disulfideOn the basis of MoS₂ as the detection platform,the improvement of traditional fluorescent probe is the focus of this chapter.The synthesis of tetraphenylethylene（TPE）molecular is cost-effective,and label-free fluorescent probe of TPE can overcome the traditional fluorescent probe such as ACQ phenomenon,light bleaching and other issues.TPE was used as a label-free fluorescent probe,while the MoS₂ nanosheet was a fluorescent quencher.Fluorescent nucleic acid biosensor was established based on the agglutination-induced emission（AIE）effect of TPE.TPE acts as a typical AIE molecule that does not emit or emits light in a dispersed solution.When DNA is added,DNA and probe are aggregated to form a complex that emits a strong fluorescent signal.However,TPE has no selectivity for either ssDNA or dsDNA.Thus,the high fluorescence quenching efficiency of the MoS₂ nanosheets and the selective adsorption of ss-and ds-DNA can be obtained toachieve label-free fluorescent nucleic acid analysis.Since this topic is only a preliminary study of a DNA biosensor based on TPE probe and MoS₂,the AIE molecule suitable for this experiment is selected based on the signal-to-noise ratio of three commonly used TPE derivatives.The fluorescence spectra prove that this method has a good feasibility.In the future study,the optimization of experimental conditions,method sensitivity and specificity,and applicability of clinical real samples will be carried out.