Synthsis Of Small Organic Molecules Luminophores Based On Energy Transfer And Their Application In Enzyme Activities Analysis

The detection and analysis of tumor markers are of great significance for the diagnosis,treatment and prognostic management of tumors.Some enzymes are biomolecules with high catalytic activity and substrate selectivity that are closely related to the growth and metabolism of tumor tissues,and their study as tumor markers has received increasing attention.For example,human NAD(P)H:quinone oxidoreductase 1(h NQO1),a flavin protease aberrantly expressed in a variety of tumor tissues,has gained extensive research as a potential tumor marker and drug target.Benefiting from the advantages of easy modification and biocompatibility,small organic molecule-based fluorescent(FL)/ electrochemiluminescent(ECL)diagnostic reagents and methods have been a hot topic of research.However,for the activity analysis of h NQO1,it is still extremely challenging to solve the problems of short emission wavelength,poor water solubility and insufficient sensitivity of small molecule fluorescent probes;secondly,the existing ECL emitters and sensing mechanisms are limited,and not sufficient to meet its application in the field of tumorrelated enzyme analysis.Thus,the development of new emitters and sensing mechanisms is important to enhance the sensing performance and promote the wide application of fluorescence and ECL technologies in the field of enzyme activity analysis.To address above issues,this thesis focuses on the design,synthesis and sensing applications of small organic molecule-based fluorescent and ECL probes based on energy transfer,which are mainly divided into the following parts.(1)In order to construct fluorescent probes based on intramolecular charge transfer for highly sensitive and selective detection of h NQO1 in vitro and activity analysis in tumor cells,this part designed and synthesized a near-infrared fluorescent probe based on the metabolic mechanism of h NQO1 and the principle of fluorescence,which effectively increased the fluorescence emission wavelength(emission wavelength of704 nm).The introduction of sulfonic acid group in the structure of probe effectively enhanced the affinity between the probe and the enzyme,and achieved a highly selective and sensitive detection of h NQO1 in vitro,obtaining a sensitivity(4.9 ng/m L)superior to that of the reported h NQO1 probe.In addition,based on the expression degree of h NQO1 in different cells,the probe enabled fluorescent imaging of tumor cells,and intracellular h NQO1 activity analysis.(2)Although fluorescent probes are effective for h NQO1 activity analysis,the mechanism of h NQO1 detection has mainly relied on a "stimulus-responsive" principle and requires the synthesis and metabolism of complex quinone-functionalized probes.However,the poor photostability and risk of hydrolysis of these fluorescent probes have severely limited the sensitivity of these methods.In this part,a novel ECL sensing system based on the endogenous "oxidation-reduction cycle" mechanism of h NQO1 was investigated,using luminol as the ECL emitter and reactive oxygen species generated from the metabolism of natural products by h NQO1 as the co-reactant,to achieve highly sensitive detection of h NQO1 and the associated assessment of antitumor drug activity.With the intrinsic cyclic amplification effect and the luminolmodified 3D nickel foam electrode,the sensing system achieved highly competitive detection limits(10 ng/m L)and signal-to-noise ratios(～460)for h NQO1.Furthermore,when immobilizing the enzyme concentration,the system was successfully applied for the rapid assessment of h NQO1-related antitumor drug activity based on the same sensing principle.This novel strategy will open a new era for h NQO1 detection and help to promote ECL as a potential tool combining the dual functions of tumor diagnosis and drug screening,facilitating the popularity of its application in protease-related tumor management.(3)The effectiveness of most ECL sensing systems for enzyme detection relies on the "co-reactant" produced by the interaction of the enzyme with the substrate.This mechanism is difficult to achieve other enzyme activity analysis that does not produce "co-reactants".In order to achieve more general ECL sensing system for enzyme analysis,the modification of the ECL emitters,which is the core component of the sensing system,is a possible approach.Unlike the conventional concept of designing ECL emitters from scratch,the paper investigates a pervasive molecular grafting scheme by synthesizing a series of luminol donor-dye acceptor based ECL emitters by grafting small molecule fluorescent dyes with diverse sensing mechanisms onto highly efficiency ECL emitters in the form of covalent bonds.The newly-emitter use resonance energy transfer(RET)as a bridge to achieve energy transfer between the excited state of the ECL emitter and the fluorescent dye,thus illuminating the ECL signal of the nonECL active dye.In addition,the newly-emitter effectively inherits the diverse response mechanisms of fluorescent dyes,such as the introduction of nitroreductase(NTR)response groups in the dye portion of the emitter,which directly catalyzes the reduction of the emitter by NTR to cause signal changes,enabling the detection of NTR.Notably,unlike the conventional two-dimensional sensing system,a new three-dimensional coordinate sensing system that can simultaneously achieve linear analysis of NTR activity and probe selectivity analysis was established for the first time by introducing chromaticity to transform the ECL spectral signal.This grafting strategy of ECL emitter preparation not only effectively expands the types and numbers of small organic molecule-based ECL emitters,but also inherits the advantages of fluorescence diversification sensing mechanism and expands the response strategy and application scenarios of ECL sensing.(4)Different from the grafting scheme based on covalent bonding interactions,the paper also utilizes non-covalent bonding interactions for molecular grafting to construct novel ECL emitters based on small molecule dyes.Stable H-CNNS/Fn complexes were prepared by simply shaking Fn and H-CNNS in a mixture of ethanol and water,and the prepared composites effectively enhanced the ECL signals of the dyes.The successful preparation of the composites was verified by structural characterization by highresolution transmission electron microscopy,infrared spectroscopy and Raman spectroscopy.The fluorescence,ECL,photo-electrochemistry and theoretical calculation properties of the complex were investigated,and it was found that the intermolecular π-π and hydrogen bonding interactions stabilized the structure of the composites,and the energy transfer between them effectively enhanced the ECL signal of fluorescein.The construction of this composites provides a paradigm for the formation of composites between nanomaterials and small molecules through noncovalent interactions,and provides an idea for the development of ECL emitters based on small molecule dyes.