| The biosensing includes the development of signal probes,recognition element and biosensing mode.The study of signal probes is an important subject of biosensing research.Cyclometalated iridium complexes have received considerable attention as photoluminescence(PL)and electrochemiluminescence(ECL)probes in the biosensing due to its high photoluminescent efficiencies,relatively long lifetimes and significant Stokes shifts in recent years.ECL biosensing has gained increasing attention in the biosensing,due to high sensitivity,high selectivity,wide linear range,controllability,and simplified optical setup.Further development of high sensitivity,high selectivity of ECL biosensing method and the expansion on the application of ECL biosensing are interesting topics in analytical chemistry.This thesis "Study on new luminescent molecules and electrochemiluminescence biosensing" Here,two respects were studied,including the designing and synthesis of new cyclometalated iridium complexes and new ECL biosensing methods.The photophysical,electrochemical,and ECL properties of eight cyclometalated iridium(Ⅲ)complexes were studied.Two PL methods based on Ir(Ⅲ)complexes were developed for the detection of two small biological molecules.Two ECL probes were developed with new recognition element.A proximity hybridization-regulated ECL bioassay with high sensitivity and high selectivity was developed for the detection of α-fetoprotein on basis of the sensitization of gold nanoparticles and target induced quenching mechanism.An ultrasensitive ECL aptasensor based on the signal amplification of graphene oxide was developed for the detection of TNF-α and secreted protein in small cell population of RAW 264.7 cells for cellular heterogeneity assessment.This thesis includes 6 chapters.Chapter 1 is general introduction,including the principle of ECL,the classification of ECL luminophores,the applications of cyclometalated iridium complexes and the applications of proximity ligation assay as well as the purpose and content of this research work.Chapter 2 Two new methods of optical chemical sensing for the determination of small biological molecules based on aldehyde bearing bis-cyclometalated Ir(Ⅲ)complex[Ir(bt)2phen-CHO]+PF6-(probe 1)and[Ir(ppy)2phen-CHO]+PF6-(probe 2)were readily prepared and their PL properities were investigated comprehensively.Both prode 1 and probe 2 diplayed weak PL due to photo induced electron transfer.Probe 1 displayed excellent selective PL enhancement in response to Hcy in neutral buffer solution based on aldehyde group cyclization reaction.The increased PL intensity of probe 1 is directly proportional to the concentration of Hcy in the range of 10 μM to 500 μM with a detection limit of 4.6 μM.Furthermore,the probe 1 has appreciable cell permeability and low cytotoxicity and has been successfully applied to selectively imaging Hcy in living cells.A nucleophilic addition reaction between HSO3-and an aldehyde group on the ancillary ligand of probe 2 occurred in the presence of HSO3-,resulting in an increase of the PL intensity of probe 2.The increase of PL signal is directly proportional to the concentration of HSO3-in the range of 0.5μM to 6 μM with a detection limit of 0.3 μM using 10 μM probe 2.More importantly,probe 2 can respond to HSO3-rapidly within 40 s.Furthermore,probe 2 was successfully applied to detect HSO3-in real white wines and the bioimaging of HSO3-in living cells.Chapter 3 Photophysical,electrochemical and electrochemiluminescence properties of 6 cyclometalated iridium complexesThe six cyclometalated iridium(Ⅲ)complexes were obtained by using dfppy,bt,and ppy as the main ligands,while avobenzone(avo),acetyl acetone(acac)and 2-(2-pyridyl)benzimidazolato-N,N’(PyBiz)as the ancillary ligands.The six cyclometalated iridium(Ⅲ)complexes were classified and numbered as probe 3-8 based on different ancillary ligands:[(dfppy)2Ir(avo)](probe 3),[(bt)2Ir(avo)](probe 4),[(ppy)2Ir(avo)](probe 5),[(ppy)2Ir(acac)](probe 6),[(bt)2Ir(acac)](probe 7),[(dfppy)2Ir(PyBiz)](probe 8),respectively.The electrochemical and ECL properties of probe 3-8 were investigated and also explained combined with density functional theory calculations.The ECL wavelength,oxidation potential and ECL efficiency were tuned by introduction of electron-withdrawing group,conjugation extent of the auxiliary ligand,different main ligands and the auxiliary ligands and different potential in the mixed system.The distinguishing of green ECL emission of the probe 8 with the red emission of Ru(bpy)32+ is based on that ECL emission peak of probe 8 is blue-shifted from that of Ru(bpy)32+ about 100 nm.Furthermore,the regulation of ECL wavelength was also realized in the same mixed solution by changing the potential.The ability to tune the luminescence of the Ir(Ⅲ)complex by changing and modifying the main or ancillary ligands makes it interesting for developing new,multicolor,light-emitting materials which are needed for full color displays and also for achieving multiple wavelength ECL labels and also makes it promising to develop ECL luminophore for multiple wavelength and potential use in multichannel analytical applications.Chapter 4 Proximity hybridization-regulated electrochemiluminescence bioassay of α-fetoprotein via target-induced quenching mechanism A proximity hybridization-regulated ECL bioassay was developed for the detection of α-fetoprotein(AFP).A biosensor was fabricated by self-assembling a capture DNA and 6-mercapto-1-hexanol via thiol-gold bond on the surface of Ru(bpy)32+/AuNPs/Nafion/GCE.After sandwich immunoassay and proximity hybridization assay among capture DNA,AFP,a pair of antibody-oligonucleotide conjugates and a signal probe(DNA-Fc),Fc in DNA-Fc was brought close to the surface of electrode in conjunction with target induced ECL quenching.The ECL intensity decreased with the increasing concentration of the AFP and AFP was monitored with a linear range of 0.05-50 ng/mL along with a detection limit of 0.04 ng/mL.The ECL bioassay is successfully applied to the detection of AFP in serum samples with one-step recognition,short operating time and good accuracy.This method displays great potential for point-of-care testing and commercial application.Chapter 5 Ultrasensitive electrochemiluminescence aptasensor for assessment of protein heterogeneity in small cell population on basis of the amplification of graphene oxide We developed an ultrasensitive ECL aptasensor for secreted protein in small cell population of RAW 264.7 cells for cellular heterogeneity assessment.An aptasensor was fabricated by self-assembly specific aptamer on gold nanoparticles modified electrode for the detection of TNF-α using sandwich assay.An ECL signal probe was prepared by combining aptamer,Ru(phen)32+,and graphene oxide(GO)through electrostatic interaction and π-π stacking interaction.The ECL intensity was linearly related to the logarithm concentrations of TNF-α in the range of 0.005-5 ng/mL with detection limit of 1.5 pg/mL.The protein heterogeneity in small cell population of RAW 264.7 cells was assessed by the ECL method.Parallel analysis was conducted to detect TNF-α secreted by RAW 264.7 cells stimulated by either lipopolysaccharides or lupeol for assessing the cell heterogeneity in small cell population and for estimating the intracellular signal transduction in inflammation.This work provides a novel approach for analysis of protein in cell secretion together with the cell heterogeneity,which is important to understand cell heterogeneity.Chapter 6 Conclusion We summarize and analyze the main conclusions and shortcomings of this thesis and provide an outlook for the further improvements and supplements.The research of this thesis provided some experimental data and references for the design and synthesis of efficient ECL signal materials and provided potential materials for the development of organic light-emitting devices.At the same time,it provided a new strategy for the design of ECL biosensors,which played an important role in promoting the development of ECL biosensing. |
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