Synthesis Of Fluorescent Probe For Copper Ion And Nitroreductase And Its Application In Biology

The fluorescence analysis method has many advantages,such as high selectivity and sensitivity,less sample dosage,simple operation,low cost and noninvasive,which is widely used in the qualitative and quantitative analysis of the cation,anion,neutral molecule,protein and DNA in the environment as well as in the organism.The Rhodamine B fluorescent probe belongs to the xanthone,which has low cost,good light stability,high molar extinction coefficient,and the advantages of both excitation and emission wavelength in visible light area.Therefore,it is widely used in the detection of all kinds of materials.1,8-anhydride naphthalene fluorophore has good planar conformation,good stability,high quantum yield and large Stocks shift.Because of their excellent photophysical and photochemical properties,these two kinds of fluorophore were chosen to design new fluorescent probe.In this paper,two kinds of Rhodamine B-based compounds RBO and RBC were successfully designed and synthesized.The RBO is a higher selective and sensitive chemosensor for Cu²⁺than other ions.It has the advantages of long emission wavelength,high selectivity,good water solubility and biocompatibility.Under the physiological conditions（p H=7.0）,the RBO lactam ring opens and releases strong fluorescence together with a color change after addition of Cu²⁺.And the ring opened in RBO to form the RBO-Cu²⁺,which was confirmed by Job’s plot,reversibility experiment and ¹H NMR.By calculation,the detection limit of RBO for Cu²⁺is down to 2.8x10^-10M,which is far lower than the standard of Cu²⁺in drinking water set by WHO.In this paper,RBO was further used to detect Cu²⁺in maize roots.It was concluded that when Cu²⁺was absorbed more than 1000μM in maize roots,Cu²⁺mainly accumulated in epidermal cells of maize roots.When Cu²⁺was less than 1000μM in maize roots,Cu²⁺mainly appeared in vascular cylinder.Therefore,this probe successfully monitored the accumulation of Cu²⁺in maize roots,which is important to understand how the plants take up,transport and store in the Cu²⁺.The excessive accumulation of Cu²⁺would restrain the activity of nitroreductase in microorganism.However,the content of nitroreductase plays a pivotal role in vivo of all microorganism normal growth.Therefore,based on 4-bromine-1,8-naphthalene anhydride,the fluorescent probe FO is designed and synthesized for detecting NTR,which is constructed by incorporating 4-nitrobenzyl bromide into 4-hydroxyl-1,8-anhydride naphthalene derivative.After strict screening,the PBS buffer solution（10 m M,p H=7,DMSO 5%）was chosen to test its in vitro property at 37°C.In the presence of NADH,the probe of FO can detect nitroreductase selectively and sensitively.And by calculation,the detection limit of FO is down to 34 ng/m L.