Novel Electrochemiluminescence System Of Luminol Derivative For Sensitive Detection Of Biomolecules

The all organs in body are made up of cells.The orderly growth and differentiation of cells can keep the health of people,but the abnormal differentiation of cells can form malignant tumor,which are often said to be cancer.Malignant tumors grow fast,easy to transfer,difficult to remove,causing serious threat to people’s life.In the early stage of malignant tumor,it shows no obvious symptoms.Therefore,the early diagnosis and prevention is the most effective means of treatment of malignant tumor.Studies have shown that excessive reactive oxygen species in cells cause oxidative stress,resulting in cell membrane lipid peroxidation and protein denaturation,then further leading to the occurrence and development of tumors.Furthermore,certain proteins secrete abnormally in the early stage of malignant tumor.Therefore,implementing the sensitive detection of these disease markers（biomolecules,proteins）has great practical value in the diagnosis and treatment evaluation of cancer.Electrochemiluminescence（ECL）is an analytical technique that combine the high controllability of electrochemical technique and the high sensitivity of luminescence technique.The ECL biosensor is a kind of biosnesors developed by combining ECL and biosnesor.It not only has the advantages of high controllability and high sensitivity,but also has excellent specificity.These excellent features make the ECL biosnesor can be used in the detection of proteins.In the study of ECL mechanism,it is found that increasing the efficiency of coreactant can improve the ECL efficiency of luminophore.In the construction process of sensor,since the immobization of luminophore determines the reaction quantity of the luminophore,effectively increasing the immobization of luminophore can improve the ECL signal of sensor.Furthermore,increasing the utilization of the target protein can also improve the ECL signal.In view of this,in order to improve the sensitivity of ECL biosensor,the paper mainly uses nanomaterials and nucleic acid amplification strategy to construct some rapid and sensitive ECL biosensors for cancer marker detection.The main contents and results are listed as follows:Part 1.Study on the construction of electrochemiluminescence immunosensor based on self-enhance N-（aminobutyl）-N-（ethylisoluminol）derivative and PdIr cubes as mimic peroxidaseAmong the signal amplification strategies,self-enhanced ECL luminophore that contain both luminescent group and coreactant group in the same molecule could greatly improve the luminescent efficiency,because the reaction distance between the luminophore and the coreactant in the same molecule is short,leading to a low energy loss.In addition,the natural peroxidase has the disadvantages of vulnerability to denaturation and difficulty of isolation.Therefore,nanomaterials with excellent stability and mimic peroxidase property have received widespread attention.In this work,PdIr cubes with efficient mimic peroxidase property were synthesized.Then,L-cysteine（L-Cys）and N-（aminobutyl）-N-（ethylisoluminol）（ABEI）were immobilized on PdIr cubes to form self-enhanced ECL nanocomplex（PdIr-L-Cys-ABEI）.Based on the self-enhanced ECL luminophore and PdIr cubes as mimic peroxidase,a simple and sensitive ECL immunosensor was fabricated and applied to detect laminin（LN）.The method provides a new way for improving the ECL efficiency of ABEI and extending its application in bioanalysis.Part 2.Study on the construction of highly sensitive electrochemiluminescence biosensor based on high-efficiency novel metal-organic framework as IndicatorThe most direct method to improve the luminous efficiency is immobization of luminophore efficiently as it determines the reaction quantity of the luminophore.The traditional method for ABEI immobilization will limit the loading amount of ABEI to a certain extent.Therefore,we proposed an efficient method for loading large amounts of ABEI.We used luminophore ABEI crosslinked 2-aminoterephhalic acid as the organic bridging ligand to synthesize Fe-MIL-101（ABEI@Fe-MIL-101）,resulting in large amounts of ABEI loaded in the skeleton structure of Fe-MIL-101.Thus,the obtained ABEI@Fe-MIL-101 exhibited excellent ECL property.Furthermore,the ECL signal was further enhanced by controlling potential to convert dissolved oxygen into superoxide radicals(O₂^˙ˉ),which avoided the use of coreagent H₂O₂ and provided a biosafe environment for protein and cell assay.The results indicated that the constructed biosensor exhibited a low detection limit of 12 cells for MUC1 on MCF-7 cancer cells,suggesting that this strategy may provide an effective method for ultrasensitive detection of biomarkers in the early diagnosis of cancer.Part 3.Study on electrochemiluminescence biosensor based on functional 3D porous conductive polymer hydrogelsReliable and sensitive in situ detection of molecule released from cells attracts tremendous research interests as it shows significance in pathological and physiological investigation.In present work,ABEI functionalized Ag nanoparticles modified three dimensional（3D）polyaniline-phytic acid conducting hydrogels（ABEI-Ag@PAni-PA）are synthesized to construct a biosensor for sensitive electrochemiluminescence（ECL）detection of hydrogen peroxide（H₂O₂）released from cells.The obtained 3D nanostructured ABEI-Ag@PAni-PA conducting hydrogels synergize the advantageous of conducting hydrogel and nanoparticle catalyst.In addition,the PAni-PA conducting hydrogels benefit the high loading density of ABEI-Ag and cell adhesion due to its 3D continuous framework,porous structure,and good biocompatibility.Importantly,compared with traditional procedure for detection of H₂O₂ released from cells that were in solution,adhesion cells on ABEI-Ag@PAni-PA conducting hydrogels provides short diffusion distance to reaction sites for H₂O₂,thus realizing sensitive in situ monitoring of H₂O₂ released from cells under drug stimulation.With good biocompatibility,high sensitivity and easy preparation,the ECL biosensor based on ABEI-Ag@PAni-PA conducting hydrogels can be expanded to detect other molecules released from cells,which may facilitate the investigation of pathological and physiological.Part 4.Study on signal-switchable electrochemiluminescence system coupled with target recycling amplification strategy for sensitive mercury ion and Mucin 1 assayRealizing the recycling of target is another effective amplification strategy to improve the signal of sensor,because a certain ECL signal could be detected in a trace level of target.In addition,studies indicated that“on-off-on”signal switch strategy has been regarded as a promising method to improve the sensitivity of sensor for it not only largely reduces the background signal but also eliminates the false positive signal.In the present work,we first found that mercury ion(Hg²⁺)has an efficient quenching effect on the ECL of ABEI,and Hg²⁺can be well immobilized via T-Hg²⁺-T interaction.Inspired by this,we fabricated an“on-off-on”ECL aptasensor based on Hg²⁺triggered signal switch for ultrasensitive determination of Hg²⁺and mucin 1.To further improve the sensitivity of the aptasensor,exonuclease I was implemented to achieve target recycling.The experimental results showed that the proposed ECL aptasensor could be used to detect Hg²⁺and MUC1 sensitively with a wide linear response.Part 5.Study on a novel electrochemiluminescence biosensor based on 3-D DNA nanomachine signal probe powered by protein-aptamer binding complexIn the traditional examination of protein,protein recycling is usually indirectly achieved by converting the target protein into single-stranded DNA by enzymatic cleavage or polymerization,making the experiment time consuming,expensive and complicated.In order to achieve the direct recycling of target protein,we constructed an ECL biosensor based on a three-dimensional（3-D）DNA nanomachine signal probe powered by protein-aptamer complex for sensitive mucin 1（MUC1）detection.In the assembly of 3-D DNA nanomachine signal probe,MUC1-aptamer complex was utilized as the catalyst of the catalytic hairpin assembly.The method could not only realize the direct recycling of the target protein,but also realize the efficient immobilization of the ABEI.In addition,CoFe₂O₄ magnetic nanomaterials was used as the nanocarrier of the 3-D DNA nanomachine signal probe,which not only can achieve rapid separation,but also catalyze the decomposition of co-reactant H₂O₂ to generate numerous reactive hydroxyl radical OH^·to realize ECL signal enhancement.The prepared ECL biosensor combined the recycling of target MUC1 and the catalysis of CoFe₂O₄,and achieved ultrasensitive detection of MUC1.Therefore,this strategy can provide an effective and sensitive detection method for clinical diagnosis,especially in trace protein determination.