Molecular Theranostics Based On Functional Nucleic Acid Amplification And Nanomaterials

Life science has a significant impact on human health and the development of modern society.Along with the constant development of life science,the combination between it with other disciplines is increasingly enhanced,which prompted the further exploration of various biological processes at the molecular level and the realization of early diseases diagnosis and treatment.In this thesis,some new methods were developed for molecular diagnosis and treatment based on functional nucleic acid amplification technology,functional nanomaterials and molecular imaging technology.Detection of disease markers adenosine-5?-triphosphate?ATP?,exosomes,glucose concentration of serum samples from diabetics,analysis of the expression level of tumor cell biomarker protein tyrosine kinase-7?PTK-7?and selective tumor therapy,construction of dual molecular imaging-guided theranostic nanoplatform were studied.Compared with traditional methods,the established analytical methods in this thesis have the advantages of simple operation,high sensitivity,high specificity,good universality and so on.The multifunctional nanoplatform can effectively realize the diagnosis and treatment of disease.The detailed contents are described as follows:In chapter 2,a fluorescence aptasensor is described that combines target-cycling strand displacement amplification?TCSDA?and s ynthesis of copper nanoclusters?Cu NCs?templated with double-stranded DNA?ds DNA?.Specifically,the detection scheme was applied to the determination of ATP via target-induced structure switching design.The binding of an aptamer hairpin probe?AP?to AT P induces a structural switch from a hairpin shape to an open conformation.This facilitates hybridization with a primer and triggers a TCSDA reaction.This amplification step produces a large quantity of ds DNA that can directly act as a template for the s ynthesis of fluorescent Cu NCs,thereby producing a strong red fluorescence?with excitation/emission maxima at 340/598 nm?that can be used to quantify ATP.The assay has a dynamic range that extends over 4 decades?from 0.01 n M to 100 n M?and a 5 p M detec tion limit.Conceivably,this detection scheme is applicable to numerous other analytes for which suitable aptamers are available.The ability to probe low-abundance biomolecules or transport a high-load drug in target cells is essential for biology and th eranostics.In chapter 3,we developed a novel activatable theranostic approach by using a structure-switching aptamer triggered hybridization chain reaction?HCR?on the cell surface,which for the first time creates an aptamer platform enabling real-time activation and amplification for fluorescence imaging and targeting therapy.The aptamer probe is designed not to initiate HCR in its free state but trigger HCR on binding to the target cell via structure switching.The HCR not only amplifies fluorescence signals from a fluorescence-quenched probe for activatable tumor imaging but also accumulates high-load prodrugs from a drug-labeled probe and induces its uptake and conversion into cisplatin in cells for selective tumor therapy.An in vitro assay shows t hat this approach affords efficient signal amplification for fluorescence detection of target PTK-7 with a detection limit of 1 p M.Live cell studies reveal that it provides high-contrast fluorescence imaging and highly sensitive detection of tumor cells,while renders high-efficiency drug delivery into tumor cells via an endocytosis pathway.The results imply the potential of the developed approach as a promising platform for early stage diagnosis and precise therapy of tumors.In chapter 4,the present work investigates the capability of single-stranded DNA?ss DNA?in enhancing the intrinsic peroxidase-like activity of the carbon nitride nanosheets?g-C₃N₄ NSs?.We found that ss DNA adsorbed on g-C₃N₄ NSs could improve the catalytic activity of the nanosheets.The maximum reaction rate of the H₂O₂-mediated TMB oxidation catalyzed by the ss DNA-NSs hybrid was at least 4times faster than that obtained with unmodified NSs.The activity enhancement could be attributed to the strong interaction between TMB and ss DNA mediated by electrostatic attraction and aromatic stacking.The high catalytic activity of the ss DNA-NSs hybrid permitted sensitive colorimetric detection of exosomes if the aptamer against CD63,a surface marker of exosome,was employed in hybrid construction.The sensor recognized the differential expression of CD63 between the exosomes produced by a breast cancer cell line?MCF-7?and a control cell line?MCF-10A?.Moreover,a similar trend was detected in the circulating exosomes isolated from the sera samples collected from breast cancer patients and healthy controls.Our work sheds lights on the possibility of using ss DNA to enhance the peroxidase-like activity of nanomaterials and demonstrates the high potential of the ss DNA-NSs hybrid in clinical diagnosis using liquid biopsy.Cobalt oxyhydroxide?CoOOH?nanoflakes,an emerging type of two-dimensional nanomaterial,show great potential for use in molecular detection.Previous assays utilizing such materials have largely been based on their outstan ding fluorescence quenching ability and oxidizing power.In chapter 5,we report the intrinsic peroxidase-like activity of cobalt oxyhydroxide?CoOOH?nanoflakes,and we show how this activity can be employed for glucose detection.We found that,in the presence of hydrogen peroxide?H₂O₂?,the nanoflakes accelerated the conversion of peroxidase substrates such as 3,3?,5,5?-tetramethylbenzidine?TMB?into colored products.By combining the CoOOH nanoflakes with the biological enzyme glucose oxidase?GOx?,we developed a colorimetric method for the detection of glucose within the concentration range 5.3-500?M.The proposed method was applied to detect elevated blood glucose levels in diabetic patients,and the intense color change induced by elevated glucose levels was found to be readily apparent to the naked eye,proving the utility of our assay for point-of-care testing.Intelligent multifunctional theranostic nanoplatforms that possess multimodality in both bioimaging and therapy are promising tools for e ffective disease battling,because they can simultaneously reduce the side-effects of therapeutic agents to normal tissues and enhance their therapeutic efficacy.In chapter 6,we developed a novel activatable imaging-guided theranostic nanoplatform using the redox-reactive nanomaterials CoOOH nanoflakes and anticancer drug Dox.This nanoplatform will be a valuable tool for magnetic resonance imaging?MRI?/optical imaging-guided drug delivery and on-demand drug release in vitro,which provides an efficient and cost effective protocol to realize successful theranostics of cancer in an intelligent and on-demand manner.The design concept can be utilized to develop more useful MRI contrast agents.This proof-of-concept design may pave the ways towards the creat ion of new nanosystems for biomedical applications based on 2D multifunctional nanomaterials.