Novel Biosensing Methods Based On Two-dimensional Nanomaterials And DNA Nanomachines

In recent years,biosensors technology as emerging technology is developed by the mutual penetration of many fields such as chemistry,biology,medicine,physics and electronic technology.Biosensors have bec ome the research focuses and been extensively applied in practical analysis and biomedicine due to the good selectivity,high sensitivity,rapid analysis speed and on-line analysis,promoting the development in clinical detection,drug screening,environmental monitoring and other fields.Taking the advantage of the low background noise,easy operation and high sensitivity,the optical measurement technology was applied in the development of biosensors,which realiz ed the highly sensitive detection of nucleic acid,metal ions and protein.Nowadays,the biosensors for the detection of the tumor markers were low-sensitive,time-consuming and false negative.In this doctoral thesis,based on two dimensional materials and DNA nanomaterials,we designed a series of highly sensitive and selective biosensors for the measurement of tumor markers and the image of cell.The detailed contents are described as follows:（1）In chapter 2,cytochrome c（Cyt c）and caspase-3 are the key mediators in apoptotic signaling.As is known to all,the release of Cyt c from mitochondria is a vital caspase activation pathway and defines the point of no-return in cell apoptosis.Here,we develop a sensitive nanosensor that holds the capability for imaging of the released Cyt c from mitochondria and caspase-3 activation cascade reaction in apoptotic signaling.The nanosensor is constructed by assembly of a fluorophore（Cy5）-tagged DNA aptamer on graphene nanosheets that have been covalently immobilized with FAM-labeled peptide.After spatially selective delivery into cytoplasm,the Cy5-tagged DNA aptamer assembled on nanosensor can bind with Cyt c released from the mitochondria to the cyt oplasm and dissociate from graphene,triggering a red fluorescence signal.In addition,the caspase-3 activated by the Cyt c released to cytoplasm can cleave the FAM-labeled peptide and result in a green fluorescence output.The nanosensor exhibits advanta ges of rapid response,high sensitivity and selectivity for in vitro assay,and high contrast imaging of Cyt c and caspase-3 in living cell.It also provides the method for the study of the kinetic relationship between the Cyt c translocation and caspase-3 activation through simultaneous imaging of Cyt c and caspase-3.The developed nanosensor described here will be an efficient and potential platform for apoptosis research.（2）In chapter 3,ascorbic acid（AA）is an important antioxidant in human body and a critical cellular metabolite involved in many biochemical pathways.Therefore,it is of great significance to monitor the AA in living cells.Nowadays,there are various technologies developed for the detection of AA,but few methods can successfully detect the intracellular AA.Here,based on ultrathin graphitic carbon nitride（g-C₃N₄）nanosheets and CoOOH nanoflakes,we reported a highly efficient biosensor（g-C₃N₄-CoOOH nanocomposite）for sensitive detection and fluorescence imaging of AA in living cell.As a fluorescence donor,the g-C₃N₄ was a promising bioimaging nanomaterial due to their high fluorescence quantum yield,good biocompatibility and low toxicity.And the CoOOH was used to be perfect fluorescence quencher.We enabled the CoOOH in situ to form a layer on the surface of g-C₃N₄,resulting in fluorescence quench of the g-C₃N₄.Upon the addition of AA,the CoOOH nanoflakes were reduced to Co²⁺,accompanying a“turn on”fluorescence signal.The g-C₃N₄-CoOOH nanocomposite exhibited highly selective response toward AA relative to other biomolecules.Moreover,this biosensor was used successfully to visualize and monitor AA in living cells.Hopefully,we believe that this biosenso r could provide a low-cost and highly sensitive platform for AA detection and bioimaging.（3）In chapter 4,DNA-based nanomachines have received increasing attention due to their great potential to mimic natural biological motors and create novel modes of motion.Here,we report a DNAzyme-based walking machine,which can operate in living cells after triggered by intracellular mi RNA-21.The walking machine is constructed by assembling DNAzyme walking strands and FAM-labeled substrate strands on a single gold nanoparticle（AuNP）.The DNAzyme walking strand is first silenced by a blocker strand.After cellular uptake,DNAzyme-based walker can be triggered by intracellular mi RNA-21 and autonomously walk along the AuNP-based 3D track fueled by DNAzyme-catalyzed substrate cleavage.Each walking step results in the cleavage of a substrate strand and the release of a FAM-labeled DNA strand,allowing real-time monitoring of the operation of the machine.The DNAzyme-based walking machine has been successfully applied to image and monitor mi RNA-21 expression levels in living cells with excellent specificity and reliability.This walking machine would hold great potential in the mi RNA associated biological research and disease diagnostics.（4）In chapter 5,DNA nanomachines are artificially designed assemblies and perform impressive movements upon stimulation by specific triggers.In this work,we report an intracellular mi RNA-initiated bipedal DNA walking machine which can real-time monitor the intracellular operation o f the walking machine and image the mi RNA-21 in living cells.This bipedal DNA walking machine is constructed with AuNPs-loaded bipedal DNAzyme walking strands silenced by the blocker strands and FAM-labeled DNA tracks.After entering into cells,intracell ular mi RNA-21 could bind to the blocker strand,releasing the DNAzyme walking strand.Subsequently,the irradiation with 302 nm light for 5 min,the leg fixed on the surface of the AuNPs would be cleaved,resulting in that the walking machine autonomously and progressively walked on the DNA tracks.Based on the continuous disassembly of substrates by DNAzyme,this autonomous and progressive walking step leaded to fluorescence signal recovery and allowed real-time monitoring of the operation of the machine.Due to the effective cleavage reaction by bipedal DNAzyme walker,this bipedal DNA walking machine outperforming other single walkers achieved ra pidly monitoring mi RNA-21expression levels with an improved sensitivity and kinetics.Therefore,this walking machine provide new insights into the application of DNA nanomachine in mi RNA associated basic biology and medical diagnosis.（5）In chapter 6,we introduce a self-powered DNA motor,which was based on DNA-functionalized gold nanoparticle（DNA-AuNP）,achieving the detection of the Bcl-2 mRNA.This DNA motor was constructed by the AuNP-based 3D tracks of FAM labeled the substrate strands and the DNA zyme strands silenced by blocker strands.The blocker strand at 3’end had a 10 nt complementary sequence with the Bcl-2 mRNA.The DNAzyme strands were designed to interact with the Bcl-2mRNA via toehold at 5’end of the DNAzyme strands,displacing DNAzyme strands from blocker strands via toehold-mediated strand displacement.Once the initiator Bcl-2 mRNA was added,the DNAmotor strand could be generated by the formation of a conjugate with a three-stranded substrate complex（blocker/Bcl-2mRNA/DNAzyme strand）.In the presence of Mn²⁺,the DNAmotor strand would cleave the FAM labeled substrate strands into two fragments,and release the FAM-labeled segment.Cleavage of the substrate provided the energy needed for the DNAzyme to move from one substrate stran d to the next,realizing the autonomous and processive walking along the AuNP.Therefore,the processive walking step achieved signal amplification for the Bcl-2 mRNA detection.