Research On Novel Fluorescent Biological Probes For Marker Detection,Early Diagnosis And Therapy Of Major Diseases

Major diseases led by cardiovascular diseases and tumors seriously threaten human health and affect their quality of life in our country due to the feature of “three-highs and one-more”,which are high prevalence,mortality,malignancy and more complications.Despite the clinical data show that early diagnosis is crucial to improve the cure rate and prognosis for these diseases,several general characteristics in the early stages of these major diseases,such as atypical symptom,less evident in imaging changes and lack of specific diagnostic markers in body fluids,make it hard to achieve early diagnosis by routine clinical examinations.Histological analysis remains the gold standard for clinical diagnosis of these major diseases.However,the commonly used pathological analysis technologies at present are still mainly based on antibody-dependent biomarker labeling technology,which are complicated-to-operate,time-consuming,and poor sensitivity and specificity for detecting early diseases.This is prone to misdiagnosis and missed diagnosis,often resulting in missing out on the best treatment period.Therefore,using analytical chemistry technical approaches to develop the novel marker detection methods,achieve early diagnosis and establish effective prevention and therapeutic measures are highly valuable for rapidly improving symptoms and avoiding exacerbations of these major diseases.The aberrant expression of some special functional molecules caused by the causative agents will lead functional and structural changes to the cells,which is the starting point for disease.Therefore,detection and functional analysis of the expression of those specific functional molecules in cells should be the fundamental method for study the molecular basis of these major diseases and achieving their early diagnosis.Due to the blocking effect of cell membrane on antibodies,the current traditional molecular biology methods mainly have to breaking the cell membrane before using the antibodies for study the expression and function of specific molecules in cells,but this operation has generally ignored intracellular environmental factors to the moleculars’ structure and function,which is not conducive to truly reflect their functions involved in many aspects of cellular life activities.Meanwhile,such methods have high requirements on the number of sample cells and are not effective for single-cell assay,which severely restricts its development in the field of precision analysis.Compared with antibody-dependent detection and imaging methods,fluorescent biological probes are favored by researchers due to the advantages of high sensitivity,fast detection speed,and no need to damage cell membranes.Especially in recent years,with the development of nanotechnology,nanoparticles represented by gold nanoparticles have been successfully used to prepare various types of nanofluorescence by their superior properties,such as free entry into cells,easy to functional modification and good biocompatibility.These probes can in situ monitor the expression changes of specific molecules in living cells and even in organelles for exploring their roles in the development and progression of diseases,and therefore show promising properties for applications in the fields of marker detection,early diagnosis and therapy of major diseases.In this dissertation,by considering several potential markers as target molecules in the early stage of major diseases,such as cardiovascular diseases and tumors,and basing on gold nanoparticles,aptamers,peptides and fluorophores,we designed and synthesized four novel fluorescent biological probes for detection and analysis of target molecules with high sensitivity and specificity,in order to explore their roles in the early stage of disease,and by this,investigating whether these molecules can be used as markers for early diagnosis of the above-mentioned major diseases,and further providing preclinical data for the effective clinical treatment of these diseases.It mainly includes the following sections:1.Based on the role of the micropeptide PLN encoded by LncRNA as a marker in the early stage of heart failure,we first developed a novel nano-fluorescent probe with an "aptamer nanoflares" structure for detection and imaging PLN in cardiomyocytes.The experimental results show that,besides the exceptional properties of excellent sensitivity,selectivity,nuclease stability and biocompatibility,compared with the traditional antibody detection methods currently used,the nanoprobe can visually monitor the dynamic changes of PLN in cardiomyocytes upon different stimulations,which is of great significance to explore the roles of PLN in the occurrence of heart failure.In addition,the nanoprobe has also been successfully applied for rapid imaging detection of PLN in myocardial tissue frozen section samples,which makes it have a good translational application prospect for clinical early diagnosis and assessment of the severity of heart failure in the future.The design concept of this probe can also be extended for imaging detection of other micropeptides in cells,and it will become a new tool to study such special sources and functional micropeptides in the future.2.Based on the role of mitochondrial Lon protease as a marker in the early stage of hypoxiainduced cardiomyocyte apoptosis,and by using the Au-Se nanoplatform developed by our group,a novel Au–Se nanoprobe with strong anti-interference capability was developed for simultaneous real-time in situ monitoring the expression of Lon protease(Lon)and Caspase-3 with high-fdelity in living cardiomyocytes.Using this nanoprobe,we verified the upstream and downstream relationship of Lon and Caspase-3 signaling molecules in hypoxia-induced apoptosis signaling pathway,and established the methods for assessing the state of cardiomyocytes under hypoxic conditions by using fluorescence intensity.In addition,the nanoprobe was also used to confirm the synergistic effect of Lon and ROS on hypoxia-induced apoptosis of cardiomyocytes and evaluate the function of ROS scavenger on attenuating such apoptosis.The applications of this nanoprobe provide theoretical support for using Lon as a diagnostic marker,and offer an alternative treatment option for early hypoxic-ischemic myocardial dysfunction.3.Since mRNA plays an important role in the body’s physiological functions,some mRNAs are also used as markers of various diseases such as tumors.We proposed a novel Au-Se bond synthesis method and developed a novel Au-Se-DNA nanoprobe named Au-Se NFs,by upgrading the Au-S bonds in nanoflares to more stable Au-Se bonds.Compared with ordinary Au-S nanoflares,The reconstructed Au-Se NFs exhibited excellent resistance to biothiol interference under physiological conditions,thus displaying a higher-fidelity fluorescent signal,and were successfully applied for accurate detection of vimentin mRNA in living breast cancer cells.With the development and application of aptamers,the specific targeting binding principle of DNA is no longer limited to simple complementary pairing between strands.Thus,this synthetic method is potentially introduced into the design of various Au-Se-DNA based nanosensors for accurate detection of various RNA,DNA and even proteins in cells,which has broad application prospects in basic research and clinical inspection work.4.Based on the role of exosomal PD-L1 in blood as an important marker in tumor immune escape and PD-1/PD-L1 immunotherapy efficacy prediction,we developed a simple,rapid and low-cost TARACET-PCR assay for the quantitative analysis of PD-L1 on exosomes that bonded to T cells via the PD-L1-PD-1 interaction with high sensitivity and selectivity.The dualtargetspecific aptamer recognition activation design of TARACET can avoid the interference from soluble forms of PD-L1 and directly measure the tumor-derived exosomal PD-L1 on T cellexosome complexes in blood samples without ultracentrifugation operation,which can truly reveal the severity of exosomal PD-L1-mediated T cell exhaustion.Therefore,with the help of large sample clinical statistics,this method is expected to be applied not only for cancer diagnosis,but also for clinical predication and medication guidance of cancers in the future.