Synthesis Of Biomolecule Modified Fluorescent Nanoclusters And Their Application For Biochemical Analysis

In recent years,fluorescent nanomaterials,especially fluorescent gold nanoclusters,have become a hot research topic worldwide.Fluorescent gold nanoclusters have been widely used in biosensing and biomedical fields owing to their simple and easy preparation,good water dispersibility,strong fluorescence signal,large Stoke displacement and good biocompatibility.In this thesis,we used moderate methods to synthesize fluorescent gold nanoclusters,for the detection of small molecules and tumor markers,which can provide an auxiliary method for clinical diagnosis and treatment.The main contents of this thesis are summarized as follows:(1)A novel ultrasensitive dual-functional biosensor for highly sensitive detection of inorganic pyrophosphate(PPi)and pyrophosphatase(PPase)activity was developed based on the fluorescent variation of globulin protected gold nanoclusters(Glo@Au NCs)with the assistance of Cu2+.Glo@Au NCs and PPi were used as the fluorescent indicator and substrate for PPase activity evaluation,respectively.In the presence of Cu2+,the fluorescence of the Glo@Au NCs will be quenched owing to the formation of Cu2+-Glo@Au NCs complex,while PPi can restore the fluorescence of the Cu2+-Glo@Au NCs complex because of its higher binding affinity with Cu2+ than that between Glo@AuNCs and Cu2+.Whereas PPase could catalyze the hydrolysis of PPi,thus released Cu2+,leading to fluorescence requenching of the.Glo@AuNCs.Based on this mechanism,quantitative evaluation of the PPi and PPase activity can be achieved ranging from 0.05 mM to 218.125 mM for PPi and from 0.1 to 8 mU for PPase,with detection limits of 0.02 mM and 0.04 mU,respectively,which is much lower than that of other PPi and PPase assay methods.More importantly,this ultrasensitive dualfunctional biosensor can also be successfully applied to evaluate the PPase activity in human serum,showing great promise for practical diagnostic applications.(2)An aptamer induced “switch on” fluorescence resonance energy transfer(FRET)biosensor for the simultaneous detection of multiple tumor markers(e.g.,AFP and CEA)combining molybdenum disulfide(MoS2)nanosheets with multicolored Au NCs by a single excitation was developed.AFP aptamer functionalized green colored Au NCs(510 nm)and CEA aptamer functionalized red colored Au NCs(650 nm)were used as energy donors,while MoS2 was used as energy receptor.On the basis of recording the change of the recovered fluorescence intensity at 510 nm and 650 nm upon the addition of targets AFP and CEA,these two tumor markers can be simultaneously quantitatively detected,with detection ranges from 0.5 ng/mL to 60 ng/m L,and 0.5 ng/m L to 120 ng/m L and detection limits of 0.16 ng/mL and 0.21 ng/m L(3?)for AFP and CEA,respectively.In addition,it is noteworthy that the developed biosensor can not only realize accurate quantitative determination of multiple tumor markers by fluorescent intensity,but also be applied in semi-quantitative determination through photo visualization.More importantly,confocal microscope experiments prove that serums from normal and hepatoma patients can also be visually and qualitatively discriminated by this FRET-based biosensor with a single excitation wavelength,indicating promising potential of this assay for clinical diagnosis.