| Enzymes and coenzymes play important roles in maintaining and regulating the physiology and metabolism of human body.Therefore,the development of technologies and methods for the accurate detecting enzymes and coenzymes in human body are necessary for clinical diagnosis and treatment.Fluorescent probes are widely used in various biological analysis.The quality of response signals of a fluorescent probe is determined by both the optical features of fluorescent materials employed in this probe and the sensing mechanism towards the target.Graphite carbon nitride(g-C3N4)nanosheets is a two-dimensional layered nanomaterial with a unique chemical structure similar to that of graphene.The high fluorescence quantum yield,the good chemical stability and biocompatibility facilitate the applications of g-C3N4 nanosheets in fabrication of fluorescent bioprobes.The research work reported in this dissertation aims at the requirements for accurate detecting coenzyme A(Co A),butyrylcholinesterase(BCh E),alkaline phosphatase(ALP),and acetylcholinesterase(ACh E)in human blood.Three novel ratiometric fluorescent and colorimetric probes based on g-C3N4 nanosheets and o-phenylenediamine(OPD)were developed,and finally a universal sensing platform was proposed by combining the above probes for sensitive and selective determination of activities of the enzymes and coenzymes mentioned above.The specific research content includes the following aspects:(1)A ratiometric fluorescent probe based on g-C3N4 nanosheets,copper ions(Cu2+)and o-phenylenediamine(OPD)was developed for detecting Coenzyme A(Co A)in human serum.The chemically inert g-C3N4nanosheets and the product of oxidation reaction of OPD(ox OPD)were employed as fluorescent indicators.Co A tends to form a complex with Cu2+,which reduces the amount of free Cu2+as both an oxidant and a fluorescence quencher in the testing system,resulting in the inhibition of OPD oxidation reaction.Since ox OPD can also quench the fluorescence emitted from g-C3N4,the ratio of the fluorescence emission intensity of g-C3N4 nanosheets to that of ox OPD(F440/F560)is facilely used as the response signal for ratiometric fluorescence detection mode.This probe exhibits linear response signals towards the concentrations of Co A in the range of 1.0–100.0?M.The detection limit(LOD,3?/S)is 0.6?M.This probe possesses good selectivity towards Co A in human blood serum,demonstrating its broad potential for detecting target in complex biological matrices.(2)A ratiometric fluorescence and colorimetric probe was developed for detecting activity of butyrylcholinesterase(BCh E)in human serum using g-C3N4nanosheets,silver ion(Ag+)and o-phenylenediamine(OPD)as chromogenic agents.Both of Ag+and the product of oxidation-reduction reaction of o-phenylenediamine(OPD)with Ag+,2,3-diaminophenazine(ox OPD),exhibited quenching ability towards the fluorescence of g-C3N4 nanosheets via photoinduced electron transfer(PET)processes.During the hydrolysis of butylthiocholine iodide(BTCh)catalyzed by BCh E,thiocholine(TCh)was generated.The rapid and strong coordination of Ag+to thiol groups of thiocholine molecules induced the significant amount decrease of Ag+in the testing system and,inhibited the oxidation of OPD,resulting in the fluorescence emission variations of both g-C3N4 nanosheets and ox OPD.Therefore,by using g-C3N4 nanosheets and ox OPD as fluorescence indicators,the intensity ratio of fluorescence emission of g-C3N4 nanosheets at 440 nm against that of ox OPD at 560 nm(F440/F560)was employed to assess the activity of BCh E.Similarly,the color variation of ox OPD indicated by the absorbance at 420 nm(?A420)was monitored for the same purpose.This dual-mode probe was validated to be sensitive and selective for detecting BCh E activity in human serum,with limits of detection(LODs)of 0.1 U L-1 for ratiometric fluorescence mode,and 0.7 U L-1 for colorimetric mode,respectively.(3)A dual-signal readout sensing platform comprising g-C3N4/Mn O2nanosheet composite and a routine chromogenic reagent o-phenylenediamine(OPD)was constructed for activity evaluation of several enzymes including Alkaline phosphatase(ALP),acetylcholinesterase(ACh E),and butyrylcholinesterase(BCh E)in human blood.Mn O2 nanosheets was employed as a catalyst for oxidizing OPD to fluorescent 2,3-diaminophenazine(ox OPD),while g-C3N4 nanosheets served as a fluorescent indicator.Mn O2 nanosheets also exhibited inner filter effect(IFE)on the fluorescence of g-C3N4 nanosheets due to the overlap of their respective absorption band and fluorescence emission band.Similarly,ox OPD would quench the fluorescence of g-C3N4 nanosheets via photoinduced electron transfer(PET)process.By using L-ascorbic acid-2-phosphate(AAP)and acetylthiocholine iodide(ATCh)as substrates,products of target enzyme-catalyzed hydrolysis reactions would reduce Mn O2 nanosheets to Mn2+cations,resulting in the inhibition of OPD oxidation and the fluorescence recovery of g-C3N4 nanosheets.Based on these reactions and effects,activities of target enzymes were indicated by the intensity ratio of fluorescence emitted from g-C3N4 nanosheets and ox OPD(F440/F560),as well as by the absorbance decrease of ox OPD at 420 nm(?A420).Limits of detection(LOD,3?/S)obtained by the sensing platform for ALP,ACh E,and BCh E were respectively0.28 U L-1,0.19 U L-1,and 0.15 U L-1 for ratiometric fluorescence mode,and 1.3 U L-1,1.0 U L-1 and 0.9 U L-1 for colorimetric mode.The sensing platform is also used for screening corresponding enzyme inhibitors and measuring the activity of target enzymes in human whole blood,with good selectivity and sensitivity.The sensing mechanisms of probes developed in this work are generally based on the combined fluorescent and color developing reagents,g-C3N4 nanosheets and OPD.It is the spectral responses of the probes towards oxidizing reagents such as metal ions or metal oxide nanomaterials that provides the signal readouts.However,these signal readouts can also reveal the amounts of targets in samples since the amounts of oxidizing reagents in the testing system are determined by the amounts targets.These sensing mechanisms make the development of universal sensing platform for detecting multi-enzymes possible.By changing the types of oxidizing reagents or substrates of enzyme-catalyzed hydrolysis,the sensing platform can be extended to detect different targets. |
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