Fluorescent Biosensors Based On Functional Nucleic Acids For Tumor Cell Imaging And Therapy

Cancer is a type of major diseases that seriously threatens human health,and its morbidity increases year by year.It is a leading cause of death worldwide.In recent decades,the diagnosis and treatment of cancer have made great progress,but there is no breakthrough in the diagnosis and treatment of cancer.Moreover,there is still no good strategy for early cancer diagnosis and treatment.At the current level of medical treatment,early detection of cancer can lead to early treatment for cancer patients,making the 5 year survival rate more than 80%,while for advanced cancer patients,the 5 year survival rate is less than 10%.Therefore,early identification and diagnosis of cancer is the prerequisite for effective cancer treatment.Meanwhile,the development of targeted therapy techniques is also an important way to reduce cancer mortality and improve the survival rate of cancer.At present,clinical diagnosis of cancer is mainly based on the imaging techniques of tissue and cell morphology,whose resolution is low.The diagnosis of cancer by these techniques can be achieved only when it has obvious occupying lesions and clinical symptoms.This makes them difficult to early diagnose cancer.After the diagnosis,the treatment of cancer is mainly based on surgical resection,chemotherapy and radiotherapy.These therapy techniques not only have limited curative effect,but also brings great pain to cancer patients.Therefore,the development of novel,rapid,highly sensitive and specific diagnostic techniques and targeted therapy methods for cancer has become an urgent task.Early diagnosis and specific targeted therapy of cancer are inseparable from specific molecular recognition and highly sensitive detection.It is well known that high sensitivity detection of tumor-related biomarkers and targeted specific recognition are effective approaches for early diagnosis,warning,and effective treatment of cancer.Specific recognition is dependent on molecular recognition elements,and efficient molecular recognition elements are the prerequisite for highly sensitive detection and specific recognition.Nucleic acids are a kind of highly efficient molecular recognition elements with the unique advantages of simple synthesis,easy modification,variable structure,and good chemical stability,and have been widely used in biological and medical research.In particular,biosensors based on nucleic acid recognition have attracted much attention in the fields of molecular detection and disease diagnosis because of their good selectivity,rapid response,low cost,and continuous dynamic monitoring in complex systems.Thus,they have developed to be the active research hotspot of analytical chemistry.However,there is no significant progress in the development of high sensitive,rapid,and real-time biosensors via the use of the unique biochemical properties of nucleic acids.Especially,the application of nucleic acidbased biosensors for imaging,detection,diagnosis and treatment of diseases in living cells and living bodies remains a great challenge.The aim of this thesis is to explore the development of novel,rapid,highly sensitive and specific biosensors for the diagnosis and treatment of cancer at the cellular levels by using the unique advantages of nucleic acid recognition.The research contents are shown as follows:1.By combining the unique fluorescence quenching property of graphene oxide（GO）and the highly specific recognition ability of aptamers,a new type of four-color fluorescent nanoprobe was constructed for the simultaneous detection and imaging of multiple tumor-related proteins in living cells.In this system,four types of aptamer probes labeled with different dyes are adsorbed onto the surface of GO through π-πstacking interaction between GO and aptamers,and forms the four-color nanoprobe.Thus,the fluorescence of the dyes is quenched by GO because of the close proximity of the dyes to the GO.When the four-color nanoprobe enters into living cells through endocytosis,the aptamer probes at the surface of GO specifically recognize and bind with intracellular tumor-related protein targets,leading to the formation and release of the aptamer-protein complexes and the recovery of the fluorescence of the corresponding dyes for simultaneous detection and imaging of multiple intracellular tumorrelated proteins.By using alpha-fetoprotein（AFP）,carcinoembryonic antigen（CEA）,vascular endothelial growth factor-165（VEGF165）and human epidermal growth factors（HER2）as model analytes,we demonstrate the principle of the developed assay method.Under the optimal assay conditions,the four-color nanoprobe can simultaneously detect these tumor related proteins in homogeneous solution with the detection limits of 61.5 pmol/L for AFP,71.2 pmol/L for VEGF165,64.5 pmol/L for CEA and 79.3 pmol/L for HER2,respectively.Results of cell imaging experiments indicate that the nanoprobe can be used to simultaneously detect and image AFP,VEGF165,CEA and HER2 in living cells,and can effectively distinguish between normal and tumor cells.Compared with the traditional methods of intracellular single protein detection,this method can obtain more comprehensive and reliable informations,effectively avoid false positive results of single marker detection,and improve the accuracy of early cancer diagnosis.2.Based on the aptamer-target specific recognition-mediate the cascade amplification of dendritic DNA,we have developed a new stimuli-responsive photodynamic imaging and therapy method,and demonstrate its application for in vitro diagnosis and treatment of cancer.This sensing system consists of five functional units: a dual functional hairpin probe（H）,two double stranded DNA substrates（FQA and FQB）,and two auxiliary DNA probes（AA and AB）.Among them,the H probe is composed of the aptamer sequences of the protein tumor marker and the initiator sequences of the DNA cascade amplification reaction;both FQA and FQB are labeled with photosensitizer Ce6 and the quench agent BHQ3.When the target is absent,the H probe is the stable state of the hairpin folding structure,which cannot trigger the amplification reaction.At this time,the fluorescence of Ce6 is in the quenching state,and it can not produce the singlet oxygen effectively under light irradiation.When the target protein was introduced into this system,the H probe can specifically bind with target protein and unfolds the hairpin structure of the H probe,leading to the initiator sequence in a free state.It crosses the substrate FQA and occurs the branching chain replacement reaction,replacing part of the substrate QA chain.Then it reacts with the auxiliary chain AA,and subsequently interacts with FQB and AB.Thus,the dendritic DNA cascade amplification reaction is initiated,and dendrimer DNA structure is generated.Finally,many FQA and FQB quenching agents are separated from Ce6,and a large number of Ce6 molecules are activated,generating strong fluorescence for detection of target protein.At the same time,activated Ce6 molecules produce large amounts of reactive oxygen species under the irradiation of 660 nm laser,which can kill cancer cells.By using CEA as a model analyte,we validate the principle of the established method.Under the optimized experimental conditions,the detection limit of CEA in homogeneous solution was as low as 15 fg/m L.Results of cell fluorescence imaging experiments indicate that this method can not only be used for ultrasensitive sensing and imaging of intracellular CEA,but also can effectively distinguish cancer cells from normal cells.In addition,because the CEA target can selectively activate numerous Ce6 molecules,the activated Ce6 molecules could produce singlet oxygen under the irradiation of 660 nm laser,so this method can selectively kill cancer cells.Compared with the reported photodynamic therapy methods,the developed stimuliresponsive photodynamic therapy method can achieve the simultaneous diagnosis and treatment of cancer,effectively avoid the phototoxicity of Ce6.Thus,it provides a new potential means for the diagnosis and accurate treatment of cancer.3.By using the unique biological properties of nucleic acids,we constructed a carrier-free dumbbell-type nucleic acid probe for the simultaneous imaging of mucin（MUC1）and telomerase activity in living cells and screening anticancer drugs.The dumbbell-type nucleic acid probe is formed by the hybridization of a hairpin DNA containing anti-MUC 1 aptamer and telomerase substrate sequences（simultaneously labeled with the fluorophore FAM and the quencher DABCYL）and a hairpin DNA containing complementary telomerase prolongation products（simultaneously labeled with the fluorophore Cy5 and the quencher BHQ3）.The dumbbell-type nucleic acid probe is stable in the absence of targets,and the fluorescence is quenched.When the targets are present,the aptamer sequences can specifically bind with MUC1 and open its hairpin structure,leading to the separation of FAM from DABCYL and restoration of the fluorescence of FAM;The telomerase can repeat the extension of the sequence of TTAGGG bases in substrate sequences,and then open the dumbbell nucleic acid probe,leading to the separation of Cy5 from BHQ3 and the recovery of the fluorescence of Cy5.Thus,it can achieve simultaneous detection of MUC1 and telomerase activity.We examined the detection performance of the dumbbell-type nucleic acid probe.The test results showed that the probe had high sensitivity and selectivity,and the detection limits obtained for MUC1 and telomerase activity were 98.68 pmol/L and 2.77×10^-8 IU/m L,respectively.Cell imaging experiments show that the probe can enter into specific cancer cells without any other carriers,simultaneously detect and image MUC1 and telomerase activity in the same cells,and effectively distinguish between MUC1-overexpressed cancer cells from other cancer cells and normal cells.In addition,the dumbbell-type nucleic acid probe also enable the in-situ screening of anticancer drugs in living cells,and evaluate the therapeutic effect of anticancer drugs in real time.This assay protocol provides a new method for cancer diagnosis and discovery of new anticancer drugs.4.By using the catalytic property of DNAzyme and the fluorescence quenching property of gold nanoparticles（Au NPs）,we designed a novel telomerase-responsive DNAzyme nanomotor for diagnosis and treatment of cancer in vitro and screening of anticancer drugs.The nanomotor system was constructed on 20 nm Au NP decorated with the DNAzyme sequence blocked by the telomerase substrate sequence and the Ce6-labeled hairpin substrate probes.When telomerase is absent,DNAzyme activity is inhibited and the DNAzyme nanomotor keeps in the closed state.In this case,the fluorescence of Ce6 is quenched because of the fluorescence quenching effect of the Au NPs,and the singlet oxygen cannot be effectively generated under light irradiation.When telomerase is introduced into the nanomotor system,the telomerase can repeat the sequences of TTAGGG in its substrate sequences,leading to the release of the extended product from Au NPs through the chain replacement reaction and the activation of the DNAzyme motor.In the presence of Mg²⁺,the activated DNAzyme can be hybridized with the Ce6-labeled hairpin substrate probe and subsequently cleave it,resulting in the release of Ce6-labeled DNA fragment and the activated DNAzyme sequences and the triggering of the fluorescence.The released DNAzyme sequences again binds with Ce6-labeled hairpin substrate probe and initiates the cyclic cleavage reaction,achieving the automatic walking of the DNAzyme sequences along Au NPs.This leads to the release of numerous Ce6 labeled DNA fragments from Au NPs,generating the significant enhancement of the fluorescence,thus achieving signal amplification for detection and imaging of telomerase activity.At the same time,the released Ce6 can generate large amounts of singlet oxygen under the irradiation of660 nm laser and selectively kill cancer cells.We evaluate the performance of DNAzyme nanomotor and the experimental results indicated that the nanomotor showed rapid response,high sensitivity and selectivity.It could detect telomerase activity with a detection limit of 9 cancer cells.Cell imaging experiments showed that the nanomotor not only can ultra-sensitively detect telomerase activity in living cells,but also can effectively distinguish between cancer cells and normal cells.It also enable in-situ screening of anticancer drugs in living cells.In addition,the DNAzyme nanomotor could effectively and selectively kill cancer cells.Compared with the reported photodynamic therapy methods,the developed DNAzyme nanomotor can significantly amplify signals for tumor imaging and achieve simultaneous diagnosis and treatment of cancer.Therefore,this DNAzyme nanomotor will provide a new way for the precise treatment of cancer.