Nitro Reductase Detection And Hypoxia Diagnosis Based On Water-soluble Multifunctional Conjugated Polymer Probes

Hypoxia is usually considered as an important feature of many diseases,such as stroke,cardiac ischemia,inflammatory,solid tumors and so on.Clinical researches show that hypoxic status of solid tumors not only leads to tumor progression toward a more malignant phenotype,but also greatly reduces the efficacy of anticancer drugs.In addition,the hypoxic status can lead to overexpression of nitroreductase(NTR)in tumor cells.Recently NTRs were used in gene-or virus-directed enzyme prodrug cancer treatment and drug screening.Thus the detection of hypoxia and nitroreductase activity plays an important role in drug researches and diagnosis of tumors.It is reported that electrochemical and fluorescent methods have been used to detect NTR level.Though the electrochemical methods possess high sensitivity and excellent detection limit,they usually need enzyme immobilization and tissue homogenate,which limit their widespread use.Interestingly,fluorescent methods attract considerable attention because they not only possess high sensitivity and selectivity,but also have spatio-temporal sampling capability,which avoid the destruction of cell structures and simplify the operation process to a great extent.In recent years,much attention was put to water-soluble conjugated polymers(WSCPs)because of their good water solubility and super-quenching properties.Therefore WSCPs are widely applied to biosensors and cell imaging.In this thesis,we designed and synthesized a novel water-soluble conjugated polymer PBFBT-NP to detect NTR sensitively.PBFBT-NP contains the nitro group and quaternary ammonium in the side chains.The introduction of quaternary ammonium makes the probe possess good water solubility.Nitro group is not only used as NTR capturing group but also can trigger PET process from the main chain to the side chain,which quenches the fluorescence of PBFBT-NP intensively.When NTRs were added to the PBFBT-NP solution,the nitro group was reduced to amino by NTRs.Simultaneously,the PET process was blocked,which induced the recovery of the fluorescence of PBFBT-NP dramatically(?110 times).The detection limit of this method was as low as 2.9 ng/mL.Furthermore,A549 tumor cells were incubated under hypoxia conditions in advance,and then were incubated with probe PBFBT-NP.The A549 cells emit strong green fluorescence,which indicates the hypoxia of cells.In addition,NTRs are abundant in E.coli bacteria.Based on specific response of PBFBT-NP to NTRs,PBFBT-NP was successfully used to replace commercial dyes for imaging E.coli cells,which not only saves costs,but also avoids the photobleaching of staining dyes.Moreover,under white light irradiation,PBFBT-NP can generate reactive oxygen species(ROS)sharply to kill E.coli.After irradiation for 60 min,about 100%of bacterial cells were dead at the PBFBT-NP concentration of 0.8?M.Cytotoxicity test shows that the multifunctional probes exhibits excellent cell compatibility.In short,the multifunctional fluorescent probe not only can be used to detect nitroreductase sensitively and selectively but also can be used to detect the degree of hypoxia.Simultaneously,this method also has great potential in the fields of anti-bacterial infection and bacteria imaging.