| Enzyme is a kind of efficient biocatalyst,which participates in the biochemical reaction of organism metabolism.The activity of enzymes in serum can specifically reflect the health of tissues or organs.Therefore,enzymes can be used as biomarkers for early diagnosis of certain diseases.Enzyme-linked immunosorbent assay(ELISA)is an important solid-phase immunoassay method,which relies on the catalytic amplification of enzymes and the specific recognition of antigens and antibodies.ELISA plays a huge role in environmental monitoring,clinical diagnosis,food safety testing,laboratory research,and biomedical applications.Research on enzyme activity sensing methods will promote the development of the ELSIA platform.Among the optical sensing methods for enzyme activity detection,colorimetry and fluorimetry are utilized more frequently.Compared with colorimetry,the fluorimetry has the virtues of simple operation,high sensitivity,fast analysis speed,and good selectivity.The construction of fluorimetry needs to select the signal unit that responds to the analyte as the analytical probe.Carbon dots exhibit superior characteristics that other conventional analytical probes do not possess,such as excellent fluorescence performance,high chemical stability,easy preparation,low toxicity,and excellent biocompatibility.In this paper,we introduced three kinds of carbon dots as analytical probes into the fluorescence sensing method for the detection of enzyme activity or the construction of ELISA platform,and further studied the application of the proposed fluorescence sensing method in actual samples.This thesis has carried out the following specific content research:1.We discovered that a mixed solution of methyldopa and ethanolamine can produce green fluorescence at room temperature.By combining UV-vis absorption spectroscopy,transmission electron microscopy,Fourier transform infrared spectroscopy,X-ray photoelectron spectroscopy,and MALDI-TOF mass spectrum analysis,we confirmed the synthesis of bright green fluorescent polymethyldopa nanoparticles(PMNPs).Inspired by such simple fluorogenic reaction and tyrosinase(TYR)-catalyzed specific conversion of metyrosine into methyldopa,we constructed a novel PMNPs-based fluorescent sensor for TYR activity detection by using metyrosine as the substrate.This fluorescence sensor has good sensitivity and selectivity to TYR in both aqueous and actual serum samples.By using kojic acid as a model,the fluorescence sensor can also be used to screen TYR inhibitors.Our research provides new prospects for TYR activity detection and screening its potential inhibitors.2.We found that green fluorescent polymer carbon dots(PCDs)can be obtained by reacting 4-aminophenol(AP)and ethylenediamine(EDA)at 37? for 2 h.Alkaline phosphatase(ALP)can hydrolyze 4-aminophenyl phosphate(APP)to generate AP,which then reacts with EDA to generate PCDs.The fluorescence intensity of PCDs is linked with the activity of ALP.Therefore,we developed a fluorescent method to detect ALP activity by generating PCDs in situ.Furthermore,we integrated this fluorescence method into the ELISA platform and developed a new type of fluorescence ELISA for the determination of cardiac troponin I(cTnI)in serum.This in situ detection method provides a new idea for the detection of biomarkers.3.We demonstrated that at room temperature,a mixed aqueous solution of N-[3-(trimethoxysilyl)propyl]ethylenediamine(DAMO)and hydroquinone(HQ)can produce fluorescent silicon-containing polymer dots(Si-PDs)within 80 min.Based on this discovery and the ability of ALP to hydrolyze sodium 4-hydroxyphenyl phosphate(4-HPP)to produce HQ,we propose a method for the detection of ALP via in situ generation of fluorescent Si-PDs.With the help of a conventional ELISA platform,we have further constructed a fluorescent ELISA for the detection of cTnI.Our research on the formation of fluorescent nanomaterials in situ has huge potential applications in ALP activity determination and disease diagnosis... |
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