Nanozymes And Lanthanide-based Luminescent Probes For Bioassays

Natural enzymes catalyze the reactions with remarkable efficiency and extraordinary specificity at mild conditions,so they have been extensively explored for various applications including biomedicine,clinic,environmental and food industry,etc.However,most of natural enzymes have several intrinsic drawbacks,such as ease-of-denaturation,ease-of-degradation,high cost,difficulty of preservation,etc.These drawbacks have in turn limited their practical applications.Therefore,the research of enzyme mimetics is vitally important.Nanomaterials possess extraordinary physicochemical properties,which enable prospective applications in advanced catalysis.They are usually considered to be inert for biological catalysis.Suprisingly,various nanomaterials,such as carbon-based nanomaterials,metal-based nanomaterials and metal oxide-based nanomaterials,etc.,have recently been found to possess catalytic activity of natural enzymes.These emerging nanomaterials with enzyme-mimic activities are collectively termed as "nanozymes".Since then,nanomaterials as a new type of promising artificial enzymes,have attracted considerable attentions.Aiming at the bottlenecks of nanozymes and combining with excellent catalytic properties of novel nanomaterials,we attempted to develop a series of nanozymes with high catalytic activities and extend the application of nanozymes for in vivo analysis.This thesis carried out the following two works regarding nanozymes:(1)Carbon-based nanomaterials as metal-free catalysts with low-cost,rich-resource and good-stability,have been extensively investigated in the field of catalytic.However,carbon-based materials have reletively lower catalytic activities compared with noble metal materials.Therefore,it is important to develop carbon-based nanozymes with superior activities for further research on the nanozymes.With heteroatoms doping of carbon-based nanomaterials,it was found that nitrogen-doped carbon materials have high peroxidase-mimic activity.Among these nitrogen-doped carbon-based nanozymes,the improvement of peroxidase-mimic activity are mainly attributed to the graphitic nitrogen.Also,it is found that these nitrogen-doped carbon-based nanozymes have similar catalytic properties to that of other nanozymes and nature horseradish peroxidase(HRP),such as the optimal pH value of peroxidase-like activity is 4,the optimal temperature under 40 ℃ and the catalytic reaction complied with the typical Michaelis-Menten equation.Nevertheless,there are no free radicals produced in the catalytic process.And these nitrogen-doped carbon-based nanozymes without multiple enzyme mimics could mimic peroxidase-like activity with high selectivity.(2)In order to further extending the application of enzymes in bioanalysis,especially in vivo,we developed a SERS biosensor with nanozymes based on gold nanoparticles’(AuNPs)peroxidase-mimic property and surface-enhanced Raman scattering(SERS)activity.In order to improve the stability of AuNPs,we synthesized metal-organic frameworks(MOFs)protected AuNPs(AuNPs@MOFs).What’s more,glucose oxidase(GOx)or lactate oxidase(LOx)were assembled onto AuNPs@MOFs to form integrative nanozymes,which exhibited good sensitivity and selectivity toward glucose and lactate detection.More,the integrative nanozymes were further explored for monitoring the concentration of glucose and lactate in living rats,which are associated with ischemic stroke and tumor.The integrative nanozymes were also used to evaluate the therapeutic efficacy of potential drugs astaxanthin(ATX)for alleviating the cerebral ischemic injuries.Compared with the traditional organic luminescent probes,lanthanide-based luminescent probes possess several distinct advantages in terms of high-efficiency luminescence,large Stokes shifts,long-lifetime luminescence,light bleaching resist,good chemical stability and low biological toxicity,which are expected to be novel luminescent probes.Although tremendous progress in bioassays have been made in the development of lanthanide-based luminescent probes,including pH,ions and biological small molecule detection.However,few studies have been devoted to saccharides detection.Heparin,a glycosaminoglycan,which has been extensively used as major injectable anticoagulants in clinical,is needed to strictly monitor its dosages and quality.Hence,it is urgent to develop effective and reliable methods for heparin detection and its quality monitoring.Combining with the importance and necessity of heparin detection,and the developed method in our lab,we constructed a de novo luminescent probe with rare earth complexes,which was applied to detect heparin with high sensitivity and selectivity,and monitor its main contaminant oversulfated chondroitin sulfate(OSCS).First,ssDNA,AG73 peptide and Tb3+ formed the biosensing system with low phosphorescence.Heparin could specifically recognize AG73 peptide and trigger the ssDNA release to sensitize the phosphorescence of Tb3+,turning on phosphorescence.So,a "turn on" phosphorescent probe for heparin was proposed,including unfractionated heparin and the low molecular weight heparin.Owing to the highly specific interaction between heparin and AG73 peptide,the established probe has high selectivity and was then successfully used for monitoring the low molecular weight heparin in metabolism in living rats.Furthermore,based on the inhibition effects of OSCS on the activity of heparinases,the proposed probe was further applied to monitor OSCS contaminant in heparin with heparinases treatment.As low as 0.002 wt%of OSCS in heparin was identified.The proposed method is simple,highly selective,which not only opens a window for polysaccharide detection,but also expands the application of lanthanide-based luminescent probes.