Analysis And Detection Of Cancer Marker Proteins Based On Membrane Interface Fluorescence Enhancement Technique Of Artificial Cell System

The traditional methods of cancer detection lack satisfactory sensitivity,accuracy and specificity in the early diagnosis of cancer,and tend to miss the optimal treatment time.However,an important challenge in the early detection of cancer is finding and validating biomarkers.The occurrence of cancers is often accompanied by the abnormal expression of several sorts of biomarkers（e.g.,nucleic acids and proteins）.Biomarker-associated proteins play an important role in the early detection of cancer.Based on the fact that artificial cells are excellent biological carriers,loading biomarkers onto the interface of cell membranes has promoted the widespread application of cancer detection.Therefore,it is urgent to develop high specificity,low detection limits and simple assays for detection of cancer-related proteins.In this thesis,several fluorescence detection and imaging techniques were developed for the rapid and specific detection of cancer-related proteins.The main contents are as follows:1.For the analysis of bioanalytes in complex matrices,it is difficult to achieve extraction and enrichment of analytes in liquid-liquid single drop microextraction system.The thesis proposed a pH-dependent polydopamine（PDA）-coated vesicles/Fe₃O₄ magnetic liquid-liquid single-drop microreactor（SDMR）for direct fluorescence detection of glutathione S-transferase（GST）,a metabolic enzyme involved with crucial biological processes in biological samples.After extracting and enriching the GST target from liquid-liquid single droplet interface,the extraction process was conducted quickly in SDMR system within 6 s.The GST was first extracted from the sample solution by Aptamer of GST（GST-Aptamer）on PDA-coated vesicles/Fe₃O₄ nanospheres（Fe₃O₄@PDA@GST-Aptamer）.Then,as the pH changes from weakly acidic to weakly basic in the SDMR system,GST and GST-Aptamer are released from Fe₃O₄@PDA@GST-Aptamer nanospheres and are captured by polydiacetylene vesicles via trapping probes.These changes alter the effective binding length and angle of the vesicle trunk,resulting in a highly enhanced fluorescence signal.This not only achieves the purpose of target enrichment,but also reduces the interference caused by matrix effect.The method can be used to directly detect GST in urine and blood without any sample pretreatment.The linear range was 0.005 to 0.5μg/m L,and the detection limit was0.834 ng/m L.The GST recovery ranges from 90.8%to 108.0%in blood samples and 91.6%to 102.8%in urine samples.By microextraction in liquid-liquid single-drop system,this method can realize the direct detection of complex samples.2.In vivo simultaneous visualization of multiple biomarkers is critical to accurately diagnose disease and decipher fundamental processes at a certain pathological evolution.In this thesis,a bi-functional aggregation-induced emission（AIE）probe was designed to detect two prostate cancer antigens.Based on the easy functionalization of aggregation-induced luminescence materials,the antibody（PSMAL）of prostate cancer membrane antigen（PSMA）and the PSA-Aptamer of free antigen（PSA）were modified with AIE probes.First,PSMA was modified on the artificial cell surface and PSA was added to simulate the real environment of prostate cancer cells.Secondly,fluorescent group Cy3 was modified on PSA-Aptamer,and PSA was captured for quantitative detection.Because of the high expression of PSMA in prostate cancer cells and its specific binding with PSMAL,the AIE probe produces strong fluorescence signal at high concentration.Under the optimal experimental conditions,the linear range for the detection of PSA and PSMA were 0.0001～0.1μg/m L,and the detection limits were 6.18 pg/m L and 8.79 pg/m L,respectively.By utilizing the photostability and anti-bleaching ability of Cy3 and AIE probes,the imaging and real-time tracking of prostate cancer cells were achieved.3.The simultaneous detection of different sorts of biomarkers will bring enormous benefits to accurate diagnosis and monitoring of the progress of diseases.In this thesis,a logical gated platform based on gold carbon dots（GCDs）loaded nucleolar protein aptamer（NCL-AS1411）was constructed and successfully applied in collaborative imaging and detection of extracellular proteins and intracellular microRNA（mi RNA）of breast cancer.In order to realize the AND logic gate operation,the magnetic beads（MB）modified with NCL-AS1411 and complementary DNA double-stranded probe（DNA-GCDs-BHQ）were magnetically separated and fixed on the surface of nanovesicles to realize signal amplification in the first step.AS1411 specifically binds to the NCL on the surface of the vesicles,and forms the G-quadruplex structure（G4）.The addition of AIE probes to interact with G4 can not only enhance its fluorescence,but also achieve the extracellular imaging NCL.Thus,the DNA-GCDs-BHQ probe on MB is detached from its complementary AS1411 and released.Due to the high biocompatibility,low toxicity and good photostability of GCDs modified at the end of the probe,the DNA probe can be transported to the nanovesicles containing mi RNA-21.The target mi RNA-21 replaces DNA-BHQ,resulting in fluorescence recovery of DNA-GCDs.By using different fluorescence characteristics of AIE and GCDs,simultaneous extracellular and intracellular detection were achieved.The method has a good linear relationship with NCL in the range of 0.005-0.5μg/m L,and the lowest detection limit is 1.176ng/m L;In the range of 0.1f M-100 p M,mi RNA-21 has a linear relationship,and the lowest detection limit is 24.47 a M.This method proposed for the first time the application of mi RNA-21 and NCL as biomarker groups for the detection of breast cancer,and realized the fluorescence quantitative detection of mi RNA-21 and NCL and breast cancer cell imaging,with good specificity and practicability.