The Establishment Of Copper Nanoclusters Fluorescent Biosensors And Its Applications In Enzyme Detection

Metal nanoclusters,emerging as a new kind of fluorescent nanomaterials,consist of several to hundreds metal atoms,and have attracted increasing attention over the past decades.The size of metal nanoclusters are down to less than 2 nm,which is equivalent to the Fermi wavelength of electrons,resulting in the discrete electronic states.Therefore,metal nanoclusters possess extraodinary properties,such as photoluminescence,excellent photostability and good biocompatibility.Metal nanoclusters were expected to be an ideal nanomaterial in environmental determination,biochemical sensing and biological imaging by exploiting these special properties.Copper is abandunt in nature and is attracting excellent attention due to its unique electric propeties.Cu NCs have also drawn considerable interest in recent years owing to the low-cost,low-toxcity and great biocompatibility.Many efforts have been devoted to the studies of Cu NCs.However,compared with gold and silver NCs,studies on Cu NCs were scarce primarily owing to their susceptibility to oxidation and difficulty in preparing extremely tiny particles.It is significant to design new strategies for synthesizing stable Cu NCs,and further enlargeing their application areas.In this paper,we studied the properties of Cu NCs synthesized in different methods and exploited the prepared Cu NCs to construct fluorescent sensors to detect several enzymes activities.The main research contents of this paper are summarized follows:1.We utilized D-penicillamine(DPA)as both the stabilizer and reductant to successfully synthsize Cu NCs.The poly(sodium-p-styrenesulfonate)(PSS)was introduced to enhance the stability of Cu NCs.The fluorescence of Cu NCs can be quenched by Cytochrome c(Cyt c)via electron transfer and electrostatic attraction between Cyt c and Cu NCs.Then trypsin can hydrolyze Cyt c to peptides and switch on the fluorescence of PSS-Cu NCs.Therefore,fluorescence“turn-off-on”detection for Cyt c and trypsin was established.The linear ranges for Cyt c detection are 8.0-40.0 n M and 40.0-680.0 n M,for trypsin,it's 0.1-6.0?g m L^-1.The human serum sample analysis indicated the reliability of the method and its potential application in real samples.2.We utilized L-Histidine as the stabilizer and ascorbic acid as the reductant to synthesize His-Cu NCs.Taking advantage of the inner filter effect between alkaline phosphatase(ALP)hydrolysis product and His-Cu NCs,we established a Cu NCs-based fluorescent probe to detect ALP activity.The linear range of ALP determination is 0.5-40.0 U/L and the detection limit is 0.045 U/L.TEM was further used to study the mechanism of fluorescence quenching.The human serum sample analysis showed promising results.3.Cu/Ag NCs were successfully prepared based on previous work and exhibited superior properties compared with copper nanoclusters.The as prepared Cu/Ag NCs were characterized by TEM?XPS?FL and FT-IR respectively.Based on the special reaction between 5,5'-dithiobis(2-nitrobenzoic acid)(DTNB)and the hydrolysis product of acetylcholinesterase(ACh E),we designed a fluorescent probe with Cu/Ag NCs for indirect detection of ACh E acticity.The linear range of ACh E is 0.1-1.0 U/L and 1.0-7.0 U/L with a detection limit of 0.03 U/L.A naked eye sensor can also be constructed owing to the color change,which brings new thoughts to the application areas.