The Application Of Nucleic Acid Nanoprobes Based On The Gold-Selenium Bond For Tumor Marker Detection

Cancer is a serious threat to human life and health,with high incidence rate and mortality.The most critical point in cancer treatment is the early diagnosis before tumor cell metastasis.Through early accurate detection of cancer,the cancer mortality can be greatly reduced.At present,the clinical detection methods of cancer,such as MRI,PET and CT,are difficult to achieve early diagnosis of cancer due to various limitations,so it is urgent to develop a more sensitive and rapid cancer detection system.Cancer markers are cancer-related biomarkers that exist in blood or other body fluids.They can be used to specifically diagnose cancer.The development of highly sensitive and specific marker detection imaging technologies is of great significance for the early and accurate diagnosis of cancer.Nanomaterials have important application value in biomedical field.Because of their unique physical and chemical properties,gold nanoparticles(Au NPs)have been used to fabricate various new chemical and biological sensors.At present,gold nanosensors based on gold-sulfur(Au-S)covalent bonds have been developed for imaging detection and treatment of various diseases.However,this traditional Au-S bond-based sensor is vulnerable to a large number of thiols in the body,which may be destroyed and replaced by free thiol molecules,which reduces the stability of the sensor,leads to distortion of the detection signal,and affects the accuracy of the results.Therefore,it is necessary to develop more reliable and stable sensors.Selenium(Se)and sulfur(S)have similar physical and chemical properties.However,compared with Au-S bond,gold-selenium(Au-Se)bond has higher bond energy and is more stable in organisms.In this thesis,based on the problem that the high thiol environment in the organism interferes with the detection signal of Au-S bond based gold nanoprobes,which is easy to cause false positive signals,a more stable Au-Se bond based nanoparticle is developed,and a series of spherical nucleic acids based on Au-Se bonds are designed and synthesized,which are used for in-situ high fidelity imaging of biomarkers in living cells and signal amplification test paper detection of cancer markers.The details are as follows:1.A spherical nucleic acid probe(SNAP)based on Au-Se bond was developed for high fidelity detection of the cancer marker mi RNA-221.This new strategy uses Au-Se bonds to replace the traditional Au-S bonds to build a more stable nanoplatform,which can effectively resist biological thiols and avoid false-positive signals.The SNAP based on Au-Se bond realized the high-fidelity detection of the tumor marker mi RNA-221 in living cells and further expanded the scope of biomarker detection.This strategy based on Au-Se bond greatly improves the stability of the probe and effectively avoids false positive signals.2.The Au-Se two-color spherical nucleic acid probe was developed.The Au-Se bond and multi-color imaging technology were introduced into the same nanoplatform to construct a more powerful and reliable dual-color Au-Se bond-based SNAP(d Se-SNAP),which was used to simultaneously detect the m RNA of two cancer markers survivin m RNA and TK1 m RNA in living cells.We successfully modified selenol derivatives to the ends of two DNA molecular beacons and assembled them to the surface of Au NPs.Compared with the dual-color Au-S bond-based SNAP(d S-SNAP),the d Se-SNAP has a higher ability to resist biological thiol interference.In addition,d Se-SNAP can effectively avoid false-positive results caused by single marker imaging,and distinguish normal cells from cancer cells by imaging two tumor markers at the same time.More importantly,the probe still has good detection performance in high thiol environment,and is more suitable for imaging in complex physiological environment.We believe that this strategy will provide more comprehensive and accurate information for cancer diagnosis and provide a more valuable perspective for the design of other sensors.3.A novel fluorescent imaging nanoprobe was designed and developed,which can sequentially detect Cyt c upstream and caspase-9 downstream in apoptosis.When the upstream Cyt c is present,the aptamer on the probe specifically binds to Cyt c and detaches from Au NPs,resulting in the recovery of Cy5 fluorescence signal coupled to the aptamer;Similarly,when the caspase-9-specific response peptide chain is cleaved by the downstream caspase-9,the cleaved peptide chain with FITC dye is detached from the Au NPs,and the FITC fluorescence signal is restored.As a control,we also synthesized a traditional nanoprobe based on Au-S bond.Compared with Au-S nanoprobes,Au-Se nanoprobes can monitor the sequential expression changes of Cyt c and caspase-9 during apoptosis,and can effectively avoid the interference of biological thiols in cells.This work provides an effective tool for accurate detection of apoptosis,and provides a new perspective for in-depth study of the relationship between disease and apoptosis.4.A point-of-care testing(POCT)system based on lateral cross flow test strip and Se-SNAP was developed to diagnose lung cancer by detecting corresponding circulating cancer mi RNAs.Compared with the traditional test paper based detection system,this POCT detection platform has two advantages:(1)the application of stable Se-SNAP can avoid false-positive signals when detecting biomarkers in a high thiol environment;(2)In the presence of the target mi RNA,a Se-SNAP can simultaneously capture multiple anti FAM antibody modified Au NPs,which makes the Au NPs aggregate in large numbers on the detection band of the test paper and generate several times amplified detection signals.The platform is simple to operate and the detection results can be distinguished by naked eyes.It is expected to be used for clinical diagnosis of lung cancer.