Synthesis Of Novel Chemiluminescent Nanoluminophores And Their Applications In Acute Myocardial Infarction Biomarker Detection

In this dissertation,the state of arts in the field of chemiluminescence(CL),CL nanoluminophores,label-free CL analysis,and in-vitro diagnostic method of acute myocardial infarction(AMI)were reviewed.Due to the simple,high sensitivity,and wide linear range,CL detection has been widely applied in bioassays.In recent years,label-free CL bioassay is a new development trend of in-vitro diagnostic techniques for the simple,rapid,and low-cost advantages.However,the developed label-free CL bioassays encounter some problems,such as insufficient sensitivity,needing coreactants,and insufficient detection accuracy,which greatly restricts their applications in in-vitro diagnosis.AMI is one of the most fatal diseases.Although diagnostic technique has been significantly improved,especially great effort has been increasingly deployed in the region of AMI diagnosis and therapy,death rate has not significantly decreased yet,mainly due to the lack of proper and rapid detection method for AMI biomarkers.Therefore,the development of a simple,rapid and low-cost label-free bioassay is of great significance for the fast diagnosis,treatment and prognosis of AMI.Due to the advantages of good biocompatibility,easy assembly,high stability and CL efficiency,CL nanoluminophore is very suitable to fabricate nano-CL interface and to develop label-free nano-CL bioassays.This dissertation focuses on the preparation of novel CL nanoluminophores and its applications in the detection of AMI biomarkers.On one hand,three kinds of AE functionalized carbon nanoluminophores with excellent CL activity were synthesized.Based on the excellent CL and special fluorescence properties of acridinium ester(AE)functionalized carbon nanoparticles nanoluminophores,a dual-mode sensing array was developed to discriminate seven kinds of transition metal ions successfully.Other the other hand,a novel nanoscale graphene oxide wrapped titanium dioxide nanoluminophore was synthesized and found to possess internal catalysis and potential-resolved ECL properties in neutral aqueous solution.In addition,Cu2+/cysteine complexes and N-(4-aminobutyl)-N-ethylisoluminol functionalized gold nanoluminophore with chitosan and nanoscale graphene oxide wrapped titanium dioxide nanoluminophore were used to fabricate nano-CL interface,which realized accurate,rapid,and sensitive detection of AMI biomarkers copeptin and cardiac troponin I(cTnl).The main results are as follows:1.AE functionalized carbon nanoparticles(CNPs),graphene oxide(GO)and multiwalled carbon nanotubes(MCNTs)nanoluminophores were successfully prepared for the first time via a simple,general and non-covalent strategy.AE were immobilized on the surface of CNPs,GO and MCNTs by electrostatic interaction,?-? interactions and amide bond.The amount of adsorbed AE on CNPs,GO and MCNTs were determined to be 3.44x10-9 mol/L,3.27x10-8 mol/L and 3.09x10-8 mol/L,respectively.The three synthesized AE functionalized carbon nanoluminophores with 5.0x10-8 mol/L of synthetic AE concentration much lower than that used for the synthesis of other CL reagents functionalized nanomaterials exhibited outstanding CL activity and stability.Carbon nanomaterials as nanosized platforms could efficiently immobilize AE and facilitate the formation of OH' and O2·-and electron transfer,leading to strong light emission.This work presented for the first time that AE functionalized nanoluminophore with outstanding CL activity could be obtained.Compared with AE,AE functionalized carbon nanoluminophores have the following advantages:(?)A chemiluminescence interface immobilizing CL reagents on a material for label-free CL bioassays is necessary.Thus,the obtained AE functionalized carbon nanomaterials with excellent CL activity may be used as nano-interface for development of label-free CL bioassays.(?)AE functionalized carbon nanoluminophores could carry a number of signal reporters so that the detection signals can be greatly amplified.And the presented simple synthesis strategy may be extended to the synthesis of other AE functionalized nanoluminophores.Additionally,the prepared AE functionalized carbon nanoparticles nanoluminophore also demonstrated excitation-dependent fluorescence properties and good fluorescence stability against photo-bleaching.Based on the excellent CL and special fluorescence properties of AE functionalized carbon nanoparticles nanoluminophore,a dual-mode sensing array was developed for the first time to discriminate seven kinds of transition metal ions successfully.2.A coreactant-free and label-free ECL immunosensor has been established for the determination of early AMI biomarker copeptin based on luminescent immuno-gold nanoassemblies consisting of Cu2+/cysteine complexes and N-(4-aminobutyl)-N-ethylisoluminol functionalized gold nanoluminophore,chitosan and immuno-gold nanoparticles.The luminescent immuno-gold nanoassemblies on the surface of indium-doped tin oxide electrode exhibited excellent ECL property,immuno-activity and good stability.In the presence of copeptin,the ECL signal decreased due to inhibition of the electron transfer by peptides.According to the decreased ECL intensity,the concentration of copeptin could be determined in range of 2.O×10-14-1.Ox10-11 mol/L with extremely low detection limit of 5.18×10-15 mol/L,which was at least 2-3 orders of magnitude lower than that of obtained by commercial enzyme linked immunosorbent assay kit and CL sandwich immunosensor.And the proposed immunosensor does not need any coreactant,and avoids complicated labeling and purification procedure.Besides,the ECL immunosensor is rapid,specific and low-cost,which can be used for the determination of copeptin in human serum samples.This work reveals that the proposed luminescent immuno-gold nanoassemblies are ideal nanointerfaces for coreactant-free and label-free immunoassays.The proposed strategy may be used for the determination of other antigens if corresponding antibodies are available.Furthermore,the coreactant-free ECL immunoassay is of great application potential in ECL immunoassays based on microfluidic devices,microchips and screen-printed electrodes.3.A novel ECL nanoluminophore,nanoscale graphene oxide(nGO)wrapped titanium dioxide nanoparticles(TiO2)(nGO@TiO2)nanoluminophore was obtained via'One-step' hydrothermal method.The prepared nGO@TiO2 nanoluminophore exhibited potential-resolved ECL emissions using K2S2O8 as coreactants in neutral aqueous solution.Two ECL peaks,ECL-1 at 510 nm and ECL-2 at 535 nm were obtained with peak potentials of-1.27 and-1.85 V,which were attributed to the ECL emission of TiO2 and nGO moiety of nGO@TiO2 nanoluminophore,respectively.In addition,by comparing the ECL intensity,we found that nGO and TiO2 in nGO@TiO2 nanoluminophore have a significant enhancement of each other's ECL emission,indicating that there is a internal catalysis effect in the nGO@TiO2 nanoluminophore.A possible ECL mechanism is proposed as follows.ECL-1 was due to that SO4·-by electro-reduction of S2O82-injected h+ into the valence band of TiO2,which recombined with electron from the conduction band to generate an light emission.In this process,nGO not only has excellent electron transfer ability but also acts as a nano-sized reaction platform to promote the generation of OH·,which could effectively promote the production of SO4·-,and thus enhance the ECL-1 emission.ECL-2 was due to reaction of electro-reduced nGO with SO4·-,which generated the excited-state of nGO,accompanying by light emission.And TiO2 could enhance the ECL emission of ECL-2 due to the excellent electron transfer ability.A novel ECL nanoluminophore with internal catalysis and potential-resolved ECL properties in neutral aqueous solution is synthesized for the first time,which provide a new idea for the design of novel and efficient ECL nanoluminophores.Furthermore,nGO@TiO2 nanoluminophore with potential-resolved ECL property might be used as ECL probe or nano-interface to realize the accurate detection of some disease biomarkers in in-vitro diagnosis.4.A label-free ECL ratiometric aptasensor was developed for the detection of cTnI in patients with AMI.nGO@TiO2 nanoluminophores with two potential-resolved ECL property were used to fabricate the ECL interface for target recognition,ECL response and amplification of signal.In the presence of cTnI,aptamer-cTnI resided from the electrode surface owing to its rigidity.The charge transfer resistance of modified working electrode reduced,resulting in the enhancement of the two ECL signals.The ECL signal of ECL-1 increased faster than that of ECL-2,which caused the increase of ECL ratio.According to the increased ECL ratio,cTnI could be detected in the range of 1.O-10?13?1.0×10-10 mol/L,which could fully meet the detection requirements of cTnI in serum of healthy people and AMI patients.Its detection limit was 3.98×1O-14 mol/L,which was at least 1 order of magnitude lower than that obtained by other reported label-free methods for the detection of cTnI.The developed label-free ECL ratio sensor has the follow advantages:(1)since the two ECL signals were derived from nGO@TiO2 nanoluminophores,it is much simpler and faster than that of ECL ratio bioassays based on labeling technology.(2)Both of nGO and TiO2 have good biocompatibility and are environment-friendly,and the detection was conducted in the neutral aqueous solution.Hence,the activity of biomolecular could be maintained well.(3)As the sensor has a dual-potential ECL emission in the negative potential range,the potential window is very narrow,and detection can be completed within 1 min.Based on the above advantages,the label-free ECL ratio biosensor is of great application potential for accurate,sensitive,rapid and specific determination of cTnI,which is of great significance for the early diagnosis and therapy of AMI patients.