| DNA nano-biointerfaces,sometimes referred to smart interfaces,are a subdivided research field with unique definition within the field of biomedicine.Generally,it refers to the conjugation products of DNA molecules with special functions and various nanomaterials by physical or chemical interactions.The macroscopic properties of DNA nano-biointerfaces can be altered by cumulative changes in microscopic interfaces in response to external stimuli.DNA-based nano-biointerfaces have abundant diversity,multitudinous unique properties,high uniformity,and reproducibility because it can fully integrate precisely controllable self-assembly and special bioactivity of DNA molecules and varied biological,physical,and chemical properties of nanomaterials.During the last ten years,as sensing module,DNA nano-biointerfaces have been widely used for the development of biosensors,which inspires a series of new concepts for designing analysis tools and tracking probes,promoting specific,sensitive,rapid,low-cost,and in situ analysis of bioactive substances.The development of DNA nano-biointerfaces for biosensors is of great significance to elucidate the molecular mechanism of life and advance the early warning and treatment of diseases.However,with continuous progression towards biology,medicine,and their interdiscipline in breadth and depth,complex biological system and processes demonstrate the functional diversity far beyond that obtained from current nanomaterials science.Also,due to the limited knowledge and technologies,a comprehensive understanding towards nano-biointerfaces cannot still be achieved.Therefore,the study on DNA nano-biointerfaces is still important direction in the many fields containing biochemical analysis in the foreseeable future.The research content of DNA nano-biointerfaces is abundant,and the work of this thesis is mainly focused on construction of novel DNA nano-biointerfaces,interfacial behavior of DNA on nanomaterials,various DNA nano-biointerfaces-based biosensing systems for molecular recognition and signal conduction,transformation,and amplification,improving biosensing capability of DNA nano-biointerfaces in complex matrices,such as serum and living cells.All of these are of significance to advance the applications of nanobiosensing techniques and biosensors in real life,such as clinical diagnosis,disease surveillance,and drug development.Protein is one of substantial basis of life activities,which is involved in various biological processes,including heredity,development,and metabolism.Protein is one of the most convenient clinical biomarker sources,when presents at increased or reduced concentrations in cells and body fluids,they can be indicative of disease stages.Therefore,in this thesis,we have constructed various biosensing systems for in situ analysis or in vitro quantification of protein biomarkers by using fabricated DNA nano-biointerfaces as sensing modules.By taking advantages of specific biorecognition of protein targets,optimizing the experimental procedures,innovating analysis concepts,we have investigated the application of single stimuli-responsive,dual stimuli-responsive and versatile DNA nano-biointerfaces in the specific protein biomarker analysis in complex matrices.The work is divided into the following parts:1.Single stimuli-responsive DNA nano-biointerfaces for quantative detection and in situ imaging of telomeraseTelomerase is a ribonucleoprotein that can maintain genome integrity by adding repetitive DNA sequences(TTAGGG)to the chromosome ends,which is tightly regulated and rarely detected in healthy somatic cells.In contrast,telomerase is found to be significantly up-regulated in 85-90%of primary tumors.This distinct characteristic makes telomerase an important indicator for early diagnosis and cancer progression.Therefore,the development of a methodology for monitoring cell-to-cell variation in telomerase activity from different samples is urgently needed in biomedical research and clinical practice.Thus,we have fabricated a conformation-switchable smart nano-biointerfaces to monitor telomerase activity based on the unique DNA extension of telomerase in living cells.The straightforward nano-biointerface contains a gold nanoparticle(Au NP)core and a dense layer of 5-carboxyfluorescein(FAM)-labeled hairpin DNA shell.The 3?-region of hairpin DNA sequence can function as the telomerase primer to be elongated in the presence of telomerase,resulting in the conformational switch of hairpin DNA and the activation of FAM.It also shows a good performance for monitoring telomerase activity in the cytoplasm by molecular imaging in cancerous cells.These advantages may offer a great potential of this method for monitoring telomerase activity in cancer progression and estimating therapeutic effect.2.Dual-responsive DNA nano-biointerfaces for in situ analysis of cytochrome c release from mitochondriaCytochrome c(Cyt c)release from mitochondria is a highly specific event,which is thought to be the point of no-return in cell apoptosis.Therefore,it is of great importance for apoptosis research via intracellular detection and in situ monitoring of Cyt c release,which cannot contribute to monitor apoptosis progression,but can also be beneficial for clearly elucidating the mechanism of apoptosis signaling.Thus,we have proposed an aptamer-based DNA nano-biointerface for the profiling of Cyt c release,which can be in response to manganese superoxide dismutase messenger RNA(Mn SOD mRNA)and Cyt c.The DNA nano-biointerface is constructed through assembling an elaborately designed Y-shaped DNA(Y-DNA)layer on gold nanoparticles(Au NPs).The fabricated assembly as a DNA nanodevice can be internalized into cytoplasm by folate-folate receptor-mediated pathway.Only if Mn SOD mRNA and Cyt c are presented,Y-DNA can be disassembled from Au NPs and thereafter outputting a fluorescence signal.So Cyt c release from mitochondria can be monitored by the DNA nanodevice.On this basis,we have proposed a new concept that an apoptotic signaling can be described with this DNA nanodevice by connected characterization of two correlative signaling molecules instead of an isolated event.Only if an apoptotic signaling pathway involving Mn SOD mRNA and downstream Cyt c is presented to serve as the input,the nanodevice can perform an?AND?logic gate operation,outputting a fluorescence signal.The concept of studying the line connecting two points will contribute to the systematic interrogation on the signaling networks in situ as the networks are composed of lines instead of individual points.And the programmable feature of this strategy also shows the potential for the profiling of other signaling pathways.3.Versatile protein-responsive DNA nano-biointerface for personal glucose meter(PGM)-based multiple protein analysis methodIf PGM,the most technology-matured point-of-care testing(POCT)device,can be properly designed for the detection of a variety of non-glucose biomarkers,it will be of great value in promoting home diagnosis of disease.Especially,considering most clinical biomarkers and therapeutic targets are proteins;thus,developing POCT of protein biomarkers with PGMs in bodily fluids is not only greatly beneficial to patients,but also helpful for promoting medical development.However,before practical application,fabrication of an efficient PGM-matching system for converting non-glucose targets into cascade glucose production remains challenging.Thus,we further explore the integration of DNA-nano-biointerface and nucleic acid signal amplification technique for highly sensitive detection of protein biomarkers.For this,we have designed a signal conversion and amplification system based on DNA-Au-Fe3O4 nano-interface,which is consisted of a catalytic hairpin assembly(CHA)reaction and a hybridization chain reaction(HCR).As a?plug-and-play?converter and amplifier,the CHA-HCR system can be immediately initiated by the detected targets,obtaining dynamic DNA assembly-directed invertase polymerization for cascade glucose production.Thus it has been successfully used for the quantification of several protein biomarkers in serum.Taking advantages of the flexible and programmable nature,the PGM-matching CHA-HCR system can be easily expanded for the POCT of other kinds of targets. |
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