Graphene-type Nanomaterials And Polypeptide Self-assemblies-based Emerging Biosensor Technology

In recent years,own to their excellent physical and chemical properties,nanomaterials have become a hot research topic,and application of nanomaterials especially inject new vitality to the rapid development of biological sensors.As the product of nanotechnology combination with biosensors,nanobiosensors was applied to many other interdisciplinary involving chemical,biological and nanoscience,providing a number of innovative ideas in the study of basic theory of the biosensor.Nanobiosensors can develop new detection principle and sensing mechanism,greatly improved its detection speed and analysis capability,possing more and more important roles in clinical testing,medical diagnosis,environmental monitoring.In this paper,combined with emerging two-dimensional nanomaterials（graphene oxide and nitride carbon graphite phase）,a series of novel biosensing strategy with simple,rapid,high sensitivity and excellent selectivity was developed,meanwhile,major disease markers（such as protein,enzyme,sugar etc.）which associated with important biological functions chose as the research target.In addition,taking advantage of peptide self-assembly technology,we proposed a novel peptide self-assembly nanoparticle for highly sensitive and selective detection of living cells,because of the poor biological compatibility and easy to obtain false positive signals employing current bioimaging probes.The main content summarized as follows:In order to further broaden the applications of two-dimensional fluorescent nanomaterials-graphite carbon nitride（g-C₃N₄）in the field of nonmetallic ions optical biosensing.In chapter 2,we report the development of a novel g-C₃N₄ nanosheet-based nanosensor strategy for highly sensitive,single-step and label-free detection of tyrosinase（TYR）activity and its inhibitor.Because of the high surface-to-volume ratio and large π-systems in the g-C₃N₄ nanosheets,as well as the catalytic oxidation of tyrosine by TYR into melanin-like polymers,the melanin-like oligomers are supposed to easily assemble on the surface,which seeds in situ growth of melanin-like polymers on the nanosheets,ultimately efficiently quenching the fluorescence of g-C₃N₄ nanosheets.This strategy was demonstrated to provide excellent selectivity and superior sensitivity and to enable rapid screening for TYR inhibitors.Therefore,the developed approach might create a useful platform for diagnostics and drugs screening for TYR-based diseases including melanoma cancer.Hydrogen peroxide（H₂O₂）and glucose are two important physical factors in the cells,Disorder or accumulation of H₂O₂ within cell can lead to the occurrence of several severe diseases such as cancer and central nervous system diseases,the blood glucose level is tightly related with hypoglycemia or diabetes,Therefore,the detection of H₂O₂ and glucose is crucial for physiology and pathology research.In chapter 3,we report for the first time that the development of a novel g-C₃N₄ nanosheets-based ratiometric fluorescence sensing strategy for highly sensitive detection of H₂O₂ and glucose.With o-phenylenediamine（OPD）oxidized by H₂O₂ in the presence of horseradish peroxidase（HRP）,the oxidization product can assemble on the g-C₃N₄ nanosheets through hydrogen bonding and π-π stacking,which effectively quenches the fluorescence of g-C₃N₄ through photoinduced electron transfer（PET）effects and delivers a new emission peak.The ratiometric signal variations enable robust and sensitive detection of H₂O₂.Based on the glucose convert into H₂O₂ through the catalysis of glucose oxidase,the g-C₃N₄-based ratiometric fluorescence sensing platform is also exploited for glucose assay.The developed strategy give a favourable selectivity detection of H₂O₂ and glucose,the detection limit is 50 n M for H₂O₂ and 0.4 μM for glucose.This strategy may provide a high-throughput platform for detecting various species involving H₂O₂-generation reactions for biomedical applications.In order to further extend the biological imaging applications of g-C₃N₄ nanosheets,in chapter 4,using the superior loading capacity of g-C₃N₄ nanosheets,we constructed a tumor targeting nanoassembly for the detection of hyaluronidase（HAase）and intracellular activatable imaging in live cells.In this strategy,a one-step approach was developed to prepare hyaluronic acid functionalized gold nanoparticles（HA-Au NPs）with HA as the template.HA acted not only as a stabilizer but also as a functional ligand for targeting cancer cell surface receptors（CD44）.Next,a noncovalent nanoassembly between g-C₃N₄ nanosheets and HA-Au NPs（i.e.,HA-Au NPs/g-C₃N₄ nanosheets）was prepared via electrostatic adsorption and hydrophobic interaction of HA-Au NPs on the surface of g-C₃N₄ nanosheets.The as-prepared nanoassembly was found to give very weak fluorescence,indicating very high fluorescence quenching efficiency for g-C₃N₄ nanosheets by HA-Au NPs via fluorescence resonance energy transfer（FRET）.The fluorescence of the nanoassembly was effectively restored in the presence of HAase,due to HA can be degraded into low molecular weight fragments by HAase,led to dismantlement of the nanoassembly.Moreover,after being transported into cells via CD44 receptor-mediated endocytosis followed by cleavage of HA by intracellular HAase,the nanoassembly provided greatly enzyme-responsive tumor-targeted imaging.Hydrogen peroxide（H₂O₂）,an important biochemical molecule,plays a pivotal role in the regulation of various physiological processes.In chapter 5,we developed a new method for using fluorophore-terminated DNA directly guided Ag NPs formation on graphene oxide surface,the as-prepared nanocomposite（Ag NPs-DNA@GO）lead to obvious fluorescence quenching of DNA by GO and Ag NPs through the resonance energy transfer.When adding H₂O₂,the DNA can be cleaved by hydroxyl radical（·OH）produced by the reaction between Ag NPs and H₂O₂,which result in disassemble Ag NPs-DNA@GO and thus achieve fluorescence enhancement.Moreover,this sensor can further extend to the glucose sensing in human serum combining with glucose oxidase（GOx）for the catalyzation of glucose and production of H₂O₂.Malignant tumor has a reputation as a deadly disease,and successful cure of most cancers depends on early detection and timely treatment.It is difficult to discover tumor in the early stage,because of the relatively low sensitivity and specificity of conventional diagnostic methods.In chapter 6,we design peptide sequence which could spontaneously assembled into elastin-like polypeptide supramolecular nanobeacon（ELPNBs）used for high sensitive and specificity cancer cells imaging.In this experiment,we have designed a set of amphiphilic elastin peptides,hydrophobic blocks which compose of Cat B-responsive peptide,labeled with fluorescent donor-acceptor pair peptide and pH-responsive histidine-rich peptide [H4V],hydrophilic segment contain MMP 2 activatable cell-penetrating peptides.Under physiological conditions,amphiphilic elastin peptides assembled into elastin-like polypeptide supramolecular nanobeacon（ELPNBs）,when ELPNBs penetrate the cell membrane by receptor-mediated endocytosis and then enter into lysosomes,which pH at 4.7-6.5,pH disrupted spherical nanoparticles,and then Cat B-responsive peptide can be cleaved to give an activatable fluorescence imaging.Exploiting cancer cell physiological environment,stepwise activatable imaging,the es tablished polypeptide supramolecular nanobeacon（ELPNBs）have achieved highly specific cancer-cell imaging.