Study On Novel Electrochemiluminescence Systems And Analytical Strategies

Electrochemiluminescence（ECL）,combined the high-controllability of electrochemical assay and high-sensitivity of chemiluminescence,has attracted great attentions due to their superior characterizations,such as low background,high sensitivity,wide dynamic range and low cost-effectiveness,which has been widely employed in both the research and industrial communities including clinical diagnostics,environmental assay and food safety,especially that associating with developments of nanotechnology and biotechnology.Now,efficient,green and multi-functionalized ECL materials were developing,while ECL assay with high sensitive,high throughput and high applicability was demonstrated.Whereas the reported ECL assays were limited in some special detection systems.It was urgently needed that ultrasensitive,simple and high applicable ECL strategies.Thus,in this thesis,some novel ECL strategies were demonstrated for sensitive detection of proteins and cells based on the self-enhanced ECL materials,multiparameter analysis and so on.This studied thesis is mainly divided in the following sections:1.Ultrasensitive cytosensor based on self-enhanced electrochemiluminescent ruthenium-silica composite nanoparticles for efficient drug screening with cell apoptosis monitoring Nowadays,it was the simple,efficient,and economical approach for cancer therapy that taking anticancer drug to regulate cells apoptosis.Currently,it was one of the efficient and reliable approaches to discover new anticancer drug and evaluate the effectiveness of potential anticancer drugs by cells apoptosis monitoring after the living cells treated with anticancer drugs.The self-enhanced ECL assay with high sensitivity could be an effective method for anticancer drug screening with cell apoptosis monitoring.Herein an ultrasensitive ECL cytosensor was reported for cell apoptosis monitoring by using self-enhanced electrochemiluminescent ruthenium-silica composite nanoparticles（Ru-N-Si NPs）labeled annexin V as signal probes.The Ru-N-Si NPs were first synthesized through simple hydrolysis of a novel precursor containing luminescent and intracoreactant groups in one molecule,which presented higher emission efficiency and enhanced ECL intensity due to the shorter electron-transfer path and less energy loss.Moreover,the as-proposed ECL cytosensor was successfully used to investigate efficiency of paclitaxel toward MDA-MB-231 breast cancer cell.The success in the establishment of the as-proposed ECL cytosensor with Ru-N-Si NPs could provide an efficient tool for anticancer drug screening by cells apoptosis monitoring and open a new way for ultrasensitive biomolecules diagnostics,cells detections and cell function researches,especially in living and complex systems.2.Multiparameter analysis-based electrochemiluminescent assay for simultaneous detection of multiple biomarker proteins on a single interface ECL assay is a powerful analytical technique with low background,high sensitivity,wide dynamic range,and cost-effectiveness,and it has attracted considerable attention in pharmaceutical analysis,clinical diagnosis,and environmental analysis.However,to the best of our knowledge,most of these established works just performed single-target detection on a single interface,which could not satisfy the demands for clinical diagnosis now,especially on the selectivity and accuracy for early diagnosis of acute diseases.It is highly valuable,but full of challenges,to develop simultaneous ECL assays for multiple detection.Herein,the multiparameter analysis of ECL-potential signals demonstrated by multivariate linear algebraic equations was first employed in the simultaneous ECL assay to realize multiple detection of biomarker proteins on a single interface.The quantitative multiparameter analysis is for the first time employed in the simultaneous ECL assay.Additionally,owing to the exponential amplification of self-synthesized nucleotide dendrimer by hybridization chain reaction（HCR）and rolling circle amplification（RCA）,the developed simultaneous ECL assay showed improved sensitivity and satisfactory accuracy for the detection of N-terminal of the prohormone brain natriuretic peptide（BNPT）and cardiac troponin I（c Tn I）.Importantly,the proposed strategy enabled simultaneous detection of multiple biomarker proteins simply,which could be readily expanded for the multiplexed estimation of various kinds of proteins and nucleotide sequence also,revealing a new avenue for early disease diagnosis with higher efficiency.3.Competitive method-based electrochemiluminescent assay with protein-nucleotide conversion for ratio detection to efficiently monitor the drug resistance of cancer cells Currently,using anticancer drugs to regulate cell apoptosis continues to remain at the front line of treatment strategies against cancer.Unfortunately,a substantial proportion of treatments with anticancer drugs are ineffective due to the drug resistance of the cancer cells via various pathways.At the early stage of drug resistance with a little increase in the expression of P-glycoprotein（P-gp）,the effectiveness of a drug could be recovered by reducing or stopping the medication.However,when the concentration of P-gp was high enough to be detected by the conventional technologies,such as western blotting and confocal laser scanning microscopy,the effectiveness of the drug couldn’t be recovered at this stage.However,the normal approaches that employ just detection of a single target were not suitable to identify the expression of P-gp in cells accurately due to the difficulty in acquiring proteins from a single cell and calculating the cell numbers.In the available P-gp detection technologies,another stably expressed protein,such as b-actin or glyceraldehyde 3-phosphate dehydrogenase（GAPDH）,was essential to be used as a control.It was a better choice to obtain these concentrations via an ECL assay using different ECL indicators on a single interface in a single run.However,inevitable limitations should be taken into consideration due to the potential cross reactions between these ECL indicators.Although the multiparameter analysis with two ECL indicators was helpful to simultaneously determine the concentration of P-gp and the stably expressed protein,such as GAPDH,to obtain the concentration ratio between these proteins,it was laborious and time-consuming.Herein,the competitive method-based ECL assay was proposed for the first time to demonstrate the concentration ratio between P-gp and GAPDH by converting these different proteins to partially coincident nucleotide sequences via a sandwich type immunoassay on magnetic beads to obtain the concentration ratio related ECL signals via competitive nucleotide hybridization on an electrode surface.The strategy was processed by a sandwich type immunoassay on magnetic beads to convert the proteins to partially coincident nucleotide sequences.Competitive nucleotide hybridization on the electrode surface would then result in an ECL signal related to the concentration ratio of the target proteins and the drug resistance of the cancer cells.The success in the establishment of the competitive method-based ECL assay offered an efficient strategy to demonstrate the concentration ratio simply.This method could be readily expanded to electrochemical,fluorescent and chemiluminescent assays for various kinds of proteins and nucleotide sequences,providing an efficient tool for early detection of drug resistance and revealing a new avenue for ultrasensitive biomolecule diagnostics,especially in cell function research.4.Adjustable ECL strategy via the target-driven circular etching on the layer-by-layer DNA micronet-carrier ECL assay as a powerful analytical technique has attracted great attentions in the sensitive determination of various targets,including metal ions,proteins,nucleotide sequences and cells,especially with the development of nanotechnology and enzyme catalytic researches.Whereas,all of these works were just suitable for the determination with fixed sensitivity and linear range due to the un-adjustable active surface of the biosensors,which could not satisfy the demands for the determination of targets in different samples.It remains a significant challenge to design ECL strategy for monitoring targets with both adjustable sensitivity and linear range.Herein,an adjustable ECL strategy was designed with detection of Pb²⁺ as a model based on the fabrication of layer-by-layer DNA microcarrier with DNAzyme and ECL indicator on the electrode surface and Pb²⁺-driven circular etching on the DNA structure to release ECL indicator,resulting decreased ECL signals associated with the reaction time and concentration of Pb²⁺.For the reaction based on DNAzyme with the Pb²⁺-driven circular cleaving,the cleaving efficiency was related with the concentration of DNAzyme（cDNAzyme）,concentration of Pb²⁺(cPb²⁺)and reaction time（t）,respectively,which was associated with the detection responses,such as ECL intensity（IntECL）.In these regards,it was possible to adjust detection performances including the sensitivity,detection limit and linear range for the detection of Pb²⁺ by adjusting detection time,providing novel insights into simple and efficient detection of Pb²⁺ with adjustable detection limit and linear range,which could be readily expanded for varies kinds of metal ions,proteins,nucleotide sequences and cells also,offering a simple and efficient technology for both environmental safe assay and clinical diagnostics.5.A sensitive immunosensor based on in situ enzymatically generating H₂O₂ to efficiently quench electrochemiluminesce of Iridium complexes doped SiO₂ nanoparticles The iridium（Ir）complexes,such as tris（2-phenylpyridine）iridium [Ir（ppy）3],exhibited longer excited-state lifetimes and higher metal-to-ligand charge transfer efficiency due to efficient intersystem crossing between the singlet and triplet excited states brought about by the strong spin-orbit coupling of the Ir metal ion.These properties were helpful to improve the detection sensitivity of ECL assay.However,low solubility in aqueous solution and high difficulty to be labeled on the bio-element limited their widely application in ECL assay.Herein,an ultrasensitive ECL assay was proposed based on nano-SiO₂ doped Ir complexes（SiO₂@Ir）as signal probes with improved ECL emission and glucose oxidase（GOD）-based enzymatic reaction to enhance the detection sensitivity via efficient quenching for Ir-based ECL emission.N-terminal of the prohormone brain natriuretic peptide（BNPT）,as efficient and widely used biomarker for acute myocardial infarction,was employed as a model to investigate its performances and application.Typically,the SiO₂@Ir in Nafion solution was coated onto the electrode surface to modify capture antibodies for BNPT based on EDC/NHS reaction via the NH2 groups on SiO₂@Ir and carboxyl groups on the antibodies.There would be an significantly improved ECL emission for SiO₂@Ir.After blocking with BSA,the prepared biosensor could be employed for the detection of BNPT via the specific antibody-antigen interaction.When there was BNPT in the detection samples,the GOD conjugated detection antibodies could be bonded onto the electrode surface.The glucose in the detection solution would be oxidized with generation of H₂O₂ via GOD-based enzymatic reaction,in which the H₂O₂ could efficiently quench the ECL emission of SiO₂@Ir.Thus,the quenched ECL emission was associated with the concentration of BNPT and could be employed to measure the it’s concentration with high sensitivity and efficiency.More importantly,the success in the preparation of ECL assay based on SiO₂@Ir and efficient quenching via enzymatic reaction could provide a simple strategy for ultrasensitive determination of biomarkers for early diagnosis and reveal a new avenue for early diagnosis of various diseases via different biomarkers,including proteins,nucleotide sequence,micro RNA and cells.