Study On Signalamplification Strategy Of Fluorescence Probe And New Methods For Biosensor

The detection of clinically relevant disease-specific biomolecules,including nucleic acids,circulating tumor cells,proteins,antibodies,and extracellular vesicles,has been indispensable to understand their functions in disease diagnosis and prognosis.Therefore,biosensors for the detection of these low-abundant biomolecules are emerging in current clinical research.Fluorescence biosensor,due to its features such as rapidness,high detection sensitivity,simplified operation and low cost,has attracted particular attention and offered new opportunities for applications in biological analysis.In this paper,by coupling the feature of high signal amplification capacity for DNA nanotechnology,a prominently stable,reproducible,and selective FL biosensors were successfully constructed and applied in targets detection.The content of this article contains following three parts:1.Versatile fluorescence detection of microRNA based on novel DNA hydrogel-amplified signal probes coupled with DNA walker amplificationIn this work,a novel DNA hydrogel-based versatile fluorescence platform combined with hybridization chain reaction（HCR）and DNA walking multiple amplification was developed for ultrasensitive detection of miRNA for the first time.A novel DNA walking machine on magnetic beads（MBs）with higher efficiency loading and significant amplification was introduced to convert target miRNA to a large number of DNA S3products.Then two different copolymer chains P1 and P2 were synthesized with acrylamide-functionalized DNA（H₂ and S₁）.With the help of DNA S₃ and H₁ on the amino functionalized SiO₂ microsphere,HCR between hairpin DNA H₂ and H₃ in P1 and P2 was initiated to assemble a novel DNA cross-linked hydrogel.After abundant SYBR Green I（SG I）were intercalated into the double-stranded DNA（dsDNA）of hydrogel or a large number of CdTe quantum dots（QDs）were labeled on the hydrogel,versatile fluorescence probes which can provide enormously amplified fluorescence signals were achieved for ultrasensitive detection of miRNAs.Taking advantages of these multiple amplification of DNA walking,HCR,DNA hydrogel,and SiO₂ microsphere,the proposed strategy demonstrated good performance with ultrahigh sensitivity for versatile assays of miRNA,and can detect miRNA-141 as low as 0.1 aM and 0.11 aM using SG I dyes and CdTe QDs fluorescent probes,respectively.Furthermore,this method can be applied to the determination of miRNA 141 in human prostate cancer with favorable precision,which indicated promising applications of the proposed sensing strategy in disease diagnosis as well as biomedical analysis.2.Ultrasensitive detection of mercury ions based on DNA dendritic macromolecular fluorescence probes combined with strand-displacement amplificationIn this work,a new kind of dendritically amplified fluorescence signal probes on SiO₂ microspheres were controllably fabricated by terminal deoxynucleotidyl transferase（TdT）-catalyzed template-free polymerization extension combined with bio-bar-code（BBC）amplification for ultrasensitive detection of Hg²⁺.In the presence of target Hg²⁺,the thymine T-Hg2+-T hairpin structure was formed and further initiated the strand-displacement-amplification（SDA）reaction,generating mimic target（MT）.MT hybridizes with capture probe 1（C1）on SiO₂ microspheres,forming a stable DNA duplex with a protruding 3’-hydroxylated sequence to initiate TdT-based DNA extension,producing abundant poly-Guanine sequence（G1）.Then G1 hybridizes with capture probes（C2）with plentiful Cytosine（C）to assemble multiple C2/reporter probe-Au nanoparticles（AuNPs）onto the SiO2 microspheres.The reporter probes further initiate TdT-based extension with poly T sequence（T1）to link large numbers of signal probes,which generate very high fluorescence signal for ultrasensitive detection of target Hg²⁺.This TdT-based signal amplification method coupled with SDA exhibits excellent selectivity and high sensitivity for Hg2+assay with a limit down to 1.0 aM.The proposed highly sensitive fluorescence strategy holds great potential for detecting targets in environmental and biological fields.3.Multifunctional fluorescence platform based on DNA walker amplification and hyperbranched DNA nanostructure for detection of multi-target and drug delivery to cancer cellsIn this work,a new hyperbranched hybridization chain reaction（HB-HCR）-based amplified fluorescence platform combined with walker-amplification was developed for versatile detection of Cu²⁺and ATP.A novel click chemistry reaction-triggered DNA walking machine on magnetic beads（MBs）was firstly introduced to convert target Cu2+to lots of DNA S3 products.With the help of DNA S3 and H1 on the amino functionalized SiO₂ microsphere,HB-HCR between hairpin HS-DNA,H3 and LT was initiated to assemble a novel dendritic DNA structure with numerous fluorescent Cy5,achieving enormously amplified signal for ultrasensitive detection of Cu²⁺.Furthermore,the dendritic structure contains large amounts of double-stranded DNA with plentiful GC bases,which can provide many loading sites for chemotherapeutic drug doxorubicin（Dox）.The specific binding of ATP to aptamer in the dendritic DNA structure allows for release of Dox,leading to activation of Dox fluorescence for ATP assay.More importantly,this dendritic DNA nanostructure loaded Dox enter into tumor cells by endocytosis,and then interacts with endogenous ATP,releasing Dox for efficient treatment of cancer cells.Taking advantages of these multiple amplification of HB-HCR on SiO2 microsphere,click chemistry reaction,DNA walking,and release of Dox,this method enabled ultrasensitive detection of Cu²⁺and ATP as low as 0.1 fM and 1 aM,respectively,which can be widely applied to multiple and accurate detection of many types of biomolecules in basic research and biomedical applications.This dendritic DNA nanostructure provided a promising tool for designing smart nanodevices for disease therapy.