Construction Of Nanomaterial-based Optical Sensors And Its Application For Enzyme Activity Evaluation

Optical sensors play an important role in the development of biosensing,owing to the advantages of low cost,simple operation,fast response speed,high sensitivity,good selectivity and so on.With the emergence of nanotechnology,optical sensing technology has been significantly developed.Compared with traditional materials,nanomaterials have unique physical and chemical properties.Since the size of the material is decreased into the nanometer level,dramatically increased surface area,surface roughness and surface area to volume ratios can be created to lead to superior physiochemical properties(i.e.,mechanical,electrical,optical,catalytic,magnetic properties,etc.).After decades of development,nanomaterials have been widely used in the field of optical sensors.At present,various nanomaterials such as manganese dioxide(MnO2)nanosheets,polydopamine nanoparticles,metal-organic frameworks(MOF),carbon quantum dots and noble metal nanoclusters have been extensively used as optical-translating units in the construction of optical sensors.By combining with the recognition unit,such as small molecular ligands,biological macromolecules and specific chemical/biological reactions,the nanomaterial-based optical sensors show specific and selective response to the target molecule.Except the function of translating sensing behavior of target into optical signals,the use of these novel nanomaterials can also endow high sensitivity to the proposed nanosensors.By taking advantage of the oxidase-like activity and quenching effect of MnO2 nanosheets,excellent fluorescence properties of fluorescent polydopamine(F-PDA)nanoparticles,and oxidase-mimicking activity of mixed valence state Ce-MOF(MVCM),we developed a series of simple,low-cost,label-free and facile synthetic biosensor for the detection of enzyme activity.Furthermore,our proposed biosensor also presented excellent applicability in human serum samples.The major content and innovation of this thesis are as follows:1.We first discovered the reversible quenching effect of MnO2 nanosheets on the fluorescence of F-PDA nanoparticles and intensively confirmed the quenching mechanism of the Forster resonance energy transfer(FRET)by using transmission electron microscopy,UV-vis spectroscopy,Fourier transform infrared spectroscopy and fluorescence lifetime experiments.With F-PDA nanoparticles serving as the fluorescent donor and MnO2 nanosheets as the fluorescent receptor to form an FRET pair,the fluorescence of F-PDA nanoparticles was efficiently quenched by the MnO2 nanosheets in the form of exponential decay.Meanwhile,alkaline phosphatase(ALP)could catalyze the hydrolysis of 2-phospho-L-ascorbic acid(AA2P)to generate L-ascorbic acid(AA),which could reduce MnO2 into Mn2+and trigger the decomposition of MnO2 nanosheets,accompanied with the fluorescence recovery of F-PDA nanoparticles.On the basis of the above reaction process,a label-free,low-cost,and specific detection of ALP was strikingly achieved.This biosensor exhibits good sensing performance for the ALP assay,with a wide linear range of 1-80 mU/mL(R2=0.999)and a low detection limit of 0.34 mU/mL as well as excellent selectivity.Moreover,the excellent applicability in human serum samples demonstrates potential applications in clinical diagnosis and biomedical research.2.Based on the oxidase-like activity of MnO2 nanosheets,we discovered that MnO2 nanosheets could directly oxidize thiamine into intensely fluorescent thiochrome.Especially,when acetylthiocholine(ATCh)and acetylcholinesterase(AChE)were introduced to this system,AChE could catalyze the hydrolysis of ATCh to generate thiocholine(TCh),whereat MnO2 nanosheets would be reduced into Mn2+ by TCh and lose the oxidase-mimicking activity,accompanied with the subsequent decrease of fluorescent intensity.However,in the presence of tacrine,the activity of the enzyme would be suppressed,which impeded the generation of TCh and destruction of MnO2 nanosheets,thus the fluorescence of this system was recovered.According to the above mechanism,we successfully established a simple,low-cost,label-free,facile and rapid synthetic fluorescent biosensor for highly sensitive detection of AChE activity and screening of its inhibitor.This fluorescent probe exhibits an excellent sensing performance,and the detection limit of AChE is as low as 15 ?U/mL,as well as the excellent specificity,sensitive inhibitor screening and real sample test were also obtained.Furthermore,such a low-toxic,good biocompatible and facile-synthesized biosensor would be promising for the potential applications in bioimaging and disease diagnosis.3.Based on the oxidase-like activity of MVCM,we found that MVCM could directly oxidize O-phenylenediamine(OPD)into 2,3-diaminophenazine(OPDox)without the participation of other oxidants.When ATCh and AChE were added into the mixed solution of MVCM and OPD,the fluorescence and absorbance intensities of MVCM-OPD system would be decreased.Since the formation of OPDox could be suppressed by TCh,which is the enzymatic hydrolysate of AChE with ATCh as the substrate.However,in the presence of tacrine,the activity of AChE would be inhibited,which impeded the generation of TCh and then promoted the formation of OPDox,thus the fluorescence of this system was recovered.According to the above mechanism,we successfully established a simple,low-toxic and label-free fluorescent and colorimetric biosensors for the detection of AChE activity and screening of its inhibitor.Our proposed biosensors showed excellent sensing performance,such as good selectivity,low detection limit(87 ?U/mL)and real sample test.These results suggested that our proposed method would be potentially applied in monitoring the disease progression.