Novel Biosensing Methods Based On Manganese Dioxide Nanoparticles And Nucleic Acid Dyes

Manganese dioxide?MnO₂?nanosheet is one of the two-dimensional nanomaterials.It has many advantages such as simple synthesis,low cost,strong fluorescence quenching ability,low toxicity and degradable by oxidation-reduction reaction.In recent years it has been widely used in the analytical detection,biological imaging,cancer treatment and so on and provides new opportunities for the treatment of various diseases and diagnosis.Nucleic acid dyes change the luminescent properties of themselves via binding to a nucleic acid.The nucleic acid dyes we often used are EB,SYBR Green I,DAPI,acridine orange?AO?,.etc,and they are often applied in the gel electrophoresis analysis,fluorescence analysis and cell imaging.In this paper,three novel fluorescent biosensors were constructed based on the manganese dioxide nanosheets and nucleic acid dyes to detect the S1 nuclease,ATP and alpha-fetoprotein,respectively.?1?In chapter 2,a novel fluorescence sensing platform for ultrasensitive detection of the S1 nuclease activity has been constructed based on MnO₂ nanosheets and FAM labeled single-stranded DNA?FAM-ssDNA?.In this system,MnO₂ nanosheets have different adsorbent ability toward ssDNA and mono-or oligonucleotide fragments.FAM-ssDNA could adsorb on MnO₂ nanosheets which resulted in significantly fluorescence quenched through fluorescence resonance energy transfer?FRET?,while mono-or oligonucleotide fragments couldn't adsorb on MnO₂ nanosheets and still retain strong fluorescence emission.With the addition of S1 nuclease,FAM-ssDNA was cleaved into mono-or oligonucleotide fragments,which weren't able to adsorb on MnO₂ nanosheets and the fluorescence signal was never quenched.The different fluorescence intensity was able to exam the S1 nuclease activity.The developed method can detect the S1 nuclease activity in the range of 0-20 U mL-1 with a detection limit of 0.05 U mL-1.Benefiting from the less time consuming and the avoiding of complex design than other endonuclease assay,a satisfactory performance for S1 nuclease in complex samples has been successfully constructed.The developed assay could potentially provide a new platform in bioimaging and clinical diagnosis.?2?In chapter 3,melamine could form hydrogen bonds with thymine?T-base?,which induced single-stranded DNA configuration transform into double-stranded DNA,and then SYBR Green I?SG I?could be embedded into double-stranded DNA to generate a strong fluorescence signal.Thus,we designed a label-free fluorescent biosensor to detect S1 nuclease and its inhibitors.When there is a 30-T base?T-30?single-stranded DNA existing in the system,a stable hydrogen bond could be formed between melamine and T-30,forming a specific double-stranded structure of T-melamine-T.Then SGI can embedded into this specific double-stranded structure,generating a stronger fluorescence signal.S1 nuclease is a single chain-specific enzyme which can hydrolyze T-30 into a single or small nucleotide fragment.In the presence of S1 nuclease,T-30 will be hydrolyzed and can not form double-stranded DNA with melamine,resulting in free SG I and weak fluorescence signal.In this way,the detection of S1 nuclease can be obtained.In the presence of ATP,the activity of S1 nuclease is inhibited and could not hydrolyze T-30.Therefore,when S1 nuclease and ATP are present simultaneously,a strong fluorescence signal can be generated and can be used to detecte ATP successfully.?3?In chapter 4,a new fluorescence biosensor based on manganese dioxide nanosheets and single-stranded DNA aptamers was designed to sensitively dectect alpha-fetoprotein.6-carboxyfluorescein-labeled single-stranded DNA?FAM-ssDNA?can be adsorbed on the surface of manganese dioxide nanosheets,then the fluorescence of FAM is quenched via fluorescence resonance energy transfer.However,FAM-ssDNA,which is the aptamer to alpha-fetoprotein,would binds to alpha-fetoprotein and could not adsorb on the surface of manganese dioxide nanosheets,leading to no fluorescence quenching and the system still maintains a strong fluorescent signal.Compared with other experimental method of detecting alpha-fetoprotein,this method is simple,rapid,time-consuming and cheap.