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Porphyrin Biomimetic Assembly-Based For Signal Probe:Application In Biochemical Biosensors

Posted on:2018-02-07Degree:DoctorType:Dissertation
Country:ChinaCandidate:P X YuanFull Text:PDF
GTID:1318330542455383Subject:Chemical Engineering and Technology
Abstract/Summary:PDF Full Text Request
Novel analytical apparatuses and methods with high sensitivity,wide versatility and user-friendliness were urgently required in the food safety,environmental protection and clinical diagnosis.The conventional biosensor normally used the enzyme as the probe label to achieve the signal transduction from biological reaction to physiological media.Unfortunately,the higher detection limit of such strategies and the unstable catalytic activity of natural enzymes,both restricted the further application of biosensor.Hence,simulating the structures of natural enzymes and searching for alternatives were of great significance to promote development of biomedical detection.Porphyrin,as the cofactors of peroxidase,heme,cytochrome and chlorophyll,with excellent photoelectric and catalytic property,have been widely used in the field of biosensors.Nowadays,great efforts have endeavored to enhance the sensitivity of those sensors.Among those,most works have been introduced to eliminate the porphyrin dimers form the simple adsorption of porphyrin and other materials.However,less attentions been on the catalytic reaction mechanism and the photoelectric properties of prophyrin.As a result,it was a meaningful work to explore the catalytic reaction principles of porphyrin and fabricate the luminescence beacon based on its photoelectric properties.In a word,the core of our proposed article was the biomimetic assembly reaction of prophyrin with other biological and nanometer materials.And then,the best biomimetic complex was introduced to enhance the signal transduction of porphyrin.Under the optimum condition,ultrasensitive bioassays based on molecular recognition was designed to realize disease diagnosis,biomedical research,and biodefense applications.This article contains five work as follow:1.The biocatalytic polymerization with the aid of HRP for surface plasmon resonance-based DNA sensingHorseradish peroxidase(HRP),a commonly natural oxidase,with encapsulate porphyrin cofactors and globulin protein,which could catalyze most biochemical reaction has drawn much attention in biosensor.A highly efficient surface plasmon resonance(SPR)-based DNA assay is developed,by employing noncovalently functionalized graphene nanosheets as the substrate,and enzymatic catalysis-induced polymerization as the mass relay.The objective of this strategy was manifold:first of all,to sensitize the overall SPRoutput by in situ optimized electrogeneration of graphene thin-film,that was characterized by atomic force micro-topography;secondly,to regulate the self-assembly and orientation of biotinylated capture probes on nickel-chelated nitrilotriacetic acid scaffolds,which were anchored onto graphene-supported pyrenyl derivatives;and lastly,to synergize the signal amplification via real-time conversion of the additive aniline into polyaniline precipitation by horseradish peroxidase-tagged reporters.With this setup,a precise and replicable DNA sensing platform for specific targets was achieved with a detection limit down to femtomolar,demonstrating a beneficial exploration and exploitation of enzymes as unique SPR signal probe for enhancement.2.Based on the bionic assembly of porphyrin and dsDNA for ultrasensitive SPR bioanalysisThis work proposed a novel label-free strategy about redox-related mass effect based on the SPRtechnique for ultrasensitive determination of DNA.The protocol starts with the modification of SPR gilded disk with the capture DNA(cDNA).After the conjugation of immobilized cDNA with the target DNA(tDNA),the hybridization chain reaction was triggered by the introduction of mutual partial complementary primers to elongate the terminal into a nanoscale duplex.As it is reported that porphyrin could intercalate into the grooves of the double-stranded DNA(dsDNA)scaffold,multiple positive-charged Fe?meso-tetra(N-methyl-4-pyridyl)porphine(FeTMPyP)with symmetric structure were uptaken for in situ formation of porphyrin-dsDNA complex.Given FeTMPyP a highly efficient catalysis for the peroxide reduction,its presence as a biomimetic cofactor was validated via circular dichroism and UV-Vis spectroscopy,demonstrating a tight binding as well as high catalytic activity and stability.Using 4-chloro-1-naphthol as a proton donor,the catalytic reduction of H2O2 would oxidize it into insoluble benzo-4-chloro-hexadienone,which simultaneously deposited on the heterogeneous interface,leading to a significant amplification in both SPR response and topological height profile.The signal increment was proportional to the concentration of tDNA,thus an ultrasensitive SPR-based DNA assay was developed with a linear range over four orders of magnitudes and a sub-femtomolar detection limit of 0.73 fM.In this work,the catalytic performance of enzyme was achieved from biomimetic assembly,demonstrating a beneficial exploitation of porphyrin as unique SPR signal probe for enhancement.3.In situ formed copper nanoparticles templated by dsDNA for enhanced SPR sensor-based DNA assayIn this work,a sensitive SPR-DNA sensor was developed by employing in situ synthesis of copper nanoparticles(CuNPs)templated by poly-T sequences DNA and synergistically with nano-effect deposition.Among this,the DNA platforms was from terminal deoxynucleotidyl transferase(TdT)-mediated extension.The objective of this strategy was manifold:firstly,tDNA hybridized with the optimal designed probes to active the TdT-mediated DNA extension onto the surface of SPR chip,resulted a long poly-T sequences ssDNA chain in dsDNA terminal onto surface of gold chip and characterized by SPR signal amplitudes.Secondly,copper ion adsorbed into the skeleton of poly-T sequences DNA,with the aid of ascorbic acid(VC)to achieve the Cu2+ reduction,copper nanostructures(CuNPs)was synchronously generated onto the single nucleotide chain anchoring in dsDNA derivatives and the formation was featured by transmission electron micrographs(TEM)and electrochemistry.Lastly,dsDNA-complexed CuNPs(CuNPs@dsDNA)triggered the final signal amplification via real-time conversion of the additive catechol violet(CV)into oligomer or chelation precipitation by CuNPs-tagged reporters.With the proposed setups,a precise and replicable DNA sensing platform for specific targets from tumor marker was obtained with a detection limit down to 3.21 femtomolar,demonstrating a beneficial of nanomaterials and multiple signal amplification.4.Carbon nitride nanosheet-supported porphyrin:a new bio mimetic catalyst for highly efficient ECL-DNA bioanalysisIn this work,a electrochemiluminescence(ECL)-based gene assay for the determination of sequence-encoding hemagglutinin of avian influenza virus type H7N9 was developed by integrating graphene-like carbon nitride(g-C3N4)nanosheet-supported cobalt proto-porphyrin IX(PPIX[Co(II)])as a tagged biomimetic catalyst.The g-C3N4 nanosheets were prepared via thermal oxidation etching of bulk graphitic C3N4 followed by liquid exfoliation.This cost-effective way produced C3N4 monolayer with large surface-to-area ratio for ?-stacking assembly of cobalt porphyrin and abundant amino groups around its edge plane for facile biofunctionalization.The delocalized electrons of g-C3N4 backbone and in-plane nitrogen-doping further provided potent axial ligation sites for tight conjugation of PPIX[Co(II)],which greatly improved its catalytic performance as mimicking enzyme.After the hybridization of target DNA with a stem-loop capture probe,the hairpin structure unfolded to expose 3'-biotinylated terminal,which could specifically recognized with the streptavidin-modified PPIX[Co(III)]@g-C3N4.Multiple PPIX[Co(III)]as highly active reduction catalysts were concentrated in the sensing interface,which drastically depleted the dissolved oxygen as endogenous coreactant of the near-infrared ECL nanoemitter 2,3-dimercaptopropane sulfonate-chelated quantum dot.The annihilated ECL intensity was inversely proportional to the concentration of target.In this work,the catalytic performance and structure of enzyme was achieved from biomimetic assembly,resulting in an ultrasensitive ECL-DNA detection.5.Red electrochemiluminescent reconstituted protein(RErP)as a universal probe for ECL detectionInspired by the chromophore of recombinant green fluorescent protein(GFP),a self-electrochemiluminescent(ECL)biomacromolecule was created in this work for early cancer detection,by the reconstitution of human apo-hemoglobin with a heme analogue:Zinc proto-porphyrin IX(ZnPPIX).A blue-shifted high-yield monochromic irradiation at 638 nm could be provokedelectrically from this red-ECL reconstituted protein(RErP)in the solution.Its stereochemistry of exogenous 10 cofactor was elucidated by circular dichroism and UV-absorption spectroscopies,while voltammetry-hyphenated surface plasmon resonance authenticated the conformational conservativeness of RErP in both stasis and electrical fields.Further conjugation with streptavidin,which process was testified by MALDI-TOF,adapted individual RErP for a universal probe of recognition specificity towards biotinylated entities.Therefore,a new enzyme-linked ECL immunoassay of vascular endothelial growth factor,a characteristic cancer biomarker,was established following a signal-on regime.On the other hand,singular porphyrin embedded in each tetrameric subunit of globin polypeptide overall accomplished a multi-tagged signal amplification without any deliberate nanofabrication.This non-photobleaching transduction nature was essentialized to be "electro-photodynamics",where the direct electrochemistry of proteins sensitized the chemiluminescent(CL)from converted singlet oxygen(1O2).Such principle realized a sensitive determination of antigen with a detection limit down to 0.74 pg·mL-1 and high precision.In this work,the performance of enzyme was changed from biomimetic assembly by porphyrin and apoHb,simplifiing the ECL detection procedure.
Keywords/Search Tags:Porphyrin, Biomimetic assembly, Biosensor, Surface plasmon resonance, Electrochemiluminescence
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