Nanomaterials And Nucleic Acid Probe-based Emerging Biosensor Technology

Biosensor is based on the identification of biological molecules and theirdownstream signaling conversion, carried out a class of analytical methods to detectthe target, crossover study is to analyze the field of chemistry and the life sciences.Compared with other analysis methods, it has many advantages, especially toimprove the speed of analysis and application flexibility. With advances inelectronics, microelectronics and mechanical systems, portable, integrateddevelopment of the sensing element, biosensing methods have the opportunity tostudy medicine, environmental protection, food security,,and public safety and otherareas to provide more on-site rapid testing technology the establishment of the firstline of defense for the public health and public safety. In recent years, researcherscontinue to make breakthroughs in the field of nano-materials, nucleic acidrecognition, nucleic acid signal amplification continuous development of newtechnologies for building high sensitivity, high selectivity, fast and efficient novelbiosensor provides perfect design ideas, as biological sensors to achieve differenttarget site rapid testing analysis provides a more powerful platform. This paper isbased on the current hot spots of nanomaterials-environmentally safe carbon, siliconnanomaterials developed three medical diagnosis application prospects biosensors,including an important cell signaling pathway protease-phospholipase D testing, livecells and subcellular organelles tracer. It’s the first reported non-graphene oxidebiosensor applied by non-covalent biological function of the first to demonstrate theuse of non-toxic nanomaterials capablely traced of mitochondria-cellular energyfactory effectively comparable organic dyes. At the same time, which involvesnanomaterials-Non-graphene oxide, silicon quantum dots, bio-functional study ofcarbon quantum dots each have a small breakthrough, laid a better foundation fortheir further development in the field of bio-sensing. The paper is also based onnucleic acid hybridization technology and metal ion-mediated nucleic acidhybridization techniques, developed detection of important cancer markers-smallRNA (microRNA) of organisms and environmental pollutants-mercury ion, and thesetwo techniques are sensitive to target to achieve a high selectivity of the analysis.All research specific contents are as follows:In order to further broaden the application of the non-oxidized graphene biosensor field, in Chapter2We developed a novel non-bilayer lipid membranesmodified graphene oxide nanocomposites, The nanoassembly can be prepared easilythrough noncovalent hydrophobic interactions between the lipid tails and thegraphene without destroying the electronic conjugation within the graphene sheet.This imparts the nanoassembly with desired electrical and optical properties withnonoxidative graphene. The phospholipid coating offers excellent biocompatibility,facile solubilization, and controlled surface modification for graphene, making thenanoassembly a useful platform for biofunctionalization of graphene. Thenanoassembly is revealed to comprise a bilayer of phospholipids with a reducedgraphene oxide sheet hosting in the hydrophobic interior, thus affording a uniqueplanar mimic of the cellular membrane. By using a fluorescein-labeled phospholipidin this nanoassembly, a fluorescence biosensor is developed for activity assay ofphospholipase D. The developed biosensor is demonstrated to have high sensitivity,wide dynamic range, and very low detection limit of0.010U/L. Moreover, becauseof its single-step homogeneous assay format it displays excellent robustness,improved assay simplicity and throughput, as well as intrinsic ability to real-timemonitor the reaction kinetics.In recent years, silicon nanomaterials are a type of important semiconductornanomaterials with attractive properties including excellentoptical/electronic/mechanical properties, favorable biocompatibility, hugesurface-to-volume ratios, surface tailorability, improved multifunctionality, as wellas their compatibility with conventional silicon technology. Silicon materials ofvarious nanostructures have been developed, among which zero-dimensionalnanomaterials-silicon quantum dots (SiQDs) have been employed as an effectivenanomaterials for various sensing applications and fluorescent biological probes forin vitro and in vivo imaging because of their unique fluorescence properties,favorable biocompatibility, noncytotoxic property, fast response and goodreproducibility. In chapter3, we report a novel kind of SiQDs which simultaneouslypossess appropriate size (approximately4nm), excellent aqueous dispersibility, highphotoluminescent quantum yield (approximately30%), and excellent photostability.In brief, fluorescent SiQDs are facilely prepared via simple one-pot electrochemicaletching method, the as-prepared SiQDs can then be passivated by hydrogen peroxideoxidation, further aging using silane-coupling reagent in the organic phase prior tothe nanoparticles transfer to the aqueous phase, then the surface of the SiQDs aremodified with carbodiimide/sulfosuccinimidyl (EDC/Sulfo-NHS) to form final hydrophilic SiQDs. The decorated SiQDs retain a certain number of primary aminegroups, may be further functionalized with functional ligands, and they arewell-suited to fluorescence imaging. In this study, we have designed and synthesizedtwo high-friendly and brightly luminescent silicon quantum dots bioconjugates,silicon quantum dots-triphenylphosphonium (SiQDs-TPP) and silicon quantumdots-morpholine (SiQDs-MP), for subcellular mitochondrial/lysosomal imaging.Two-photon confocal imaging experiments establish that SiQDs-TPP and SiQDs-MPwere able to accurately and quickly localize to mitochondrias and lysosomesrespectively (approximately15min). Colocalization studies of the probe withcommercial colocalization reagents in cells demonstrated the specific localization ofthe probe in the mitochondrias or lysosomes with an extremely high colocalizationefficiency. Furthermore, SiQDs-TPP possesses high specificity to mitochondria,superior photostability, and appreciable tolerance to environmental change, allowingimaging and tracking of the mitochondrial morphological changes in a long period oftime, in early apoptosis, for example, swelling of mitochondria are clearly foundwhen the cells were treated with staurosporine(STS), a commonly used apoptosisinducer. The new imaging probe has a number of properties that far exceed those ofcommercial colocalization probes, including high mitochondrial/lysosomalselectivity, good fluorescence quantum yield, and, importantly, high photostability,high-friendly, all resulting in a superior figure of merit.As the classical graphene, carbon-based nanomaterial offer several favorableattributes including excellent optical/electrical properties, non-toxic, chemicallyinert, easy metabolism and promise significant advantages in several imaging andbioanalytical applications. The aim of this work is to further extend the applicationof fluorescent carbon nanomaterials in the field of biochemistry to develop anfluorescent biological probes for cell imaging. In Chapter4, we synthesized a novelkind of carbon nanodots (C-dots), which specifically involves decorating thenanoparticles surface with liposome bilayer. Current synthetic methods of C-dots aremainly deficient in accurate control of uniform dimensions and the resulting surfacechemistry, as well as in obtaining fluorescent materials with low quantum yields(usually below3%). Most of these synthesis methods need post treatment withsurface passivating agents in order to improve their water solubility andluminescence property. Recently, for example, serious efforts have been made tosyntheses of amino-functionalized fluorescent C-dots (quantum yields wasapproximately10%) by amino-modified PEG (MW=1500). On the basis of the above, we first synthesis carbon precursor through conventional nitric acid reflux method,and obtain required uniform particle size (less than10nm) by ultrafiltration, andfurther tried a similar molecular weight phospholipids instead of amino-modifiedPEG for carbon surface passivation. The final C-dots were obtained via specialcoordination crosslinked interaction between dipalmitoyl phosphatidylethanolamine(DPPE) of the as-prepared liposome decorated C-dots and carboxyl functionalgroups that present in the surface of C-dots due to oxidation using the EDC andsulfo-NHS. The as-prepared liposome decorated C-dots have a uniform dispersionwithout apparent aggregation and particle diameters of5nm, excellentbiocompatibility, controllable surface modification and their fluorescence intensitythree times than carbon precursor at same concentration. We further explored thetracking and distribution of as-prepared liposome decorated C-dots in cells, Asexpected, after incubating with a C-dots-culture solution, two-photon confocalimaging experiments establish that the fluorescence in the Hela cells retained a highintensity, and distributed in various locations. This indicating the as-preparedliposome decorated C-dots ideally suited for applications in the field of biologicalimaging.Mercury is highly toxic global environmental pollutants, especially its highmobility, persistence, the role of methylation, bioaccumulation and amplification ofthe characteristics of the food chain, even a very small amount present in theenvironment, for plants, animals and human health is also a great threat. In Chapter5, we have designed a very novel, based on T-Hg2+-T coordination chemistry, energysensitivity, high selectivity to detect Hg2+. This strategy exploited the cooperativityof proximate poly-T oligonucleotides in coordination with Hg2+. Ferrocene(Fc)-tagged poly-T oligonucleotides were immobilized on the electrode surface viaself-assembly of the terminal thiol moiety. In the presence of Hg2+, a pair of poly-Toligonucleotides could cooperatively coordinate with Hg2+, which triggered aconformational reorganization of the poly-T oligonucleotides from flexible singlestrands to relatively rigid duplexlike complexes, thus drawing the Fc tags away fromthe electrode with a substantially decreased redox current. The responsecharacteristics of the sensor were thoroughly investigated using capillaryelectrophoresis and electrochemical measurements. The results revealed that thesensor showed a sensitive response to Hg2+in a concentration range from1.0nM to2.0μM, with a detection limit of0.5nM. Also, this strategy afforded exquisiteselectivity for Hg2+against a reservoir of other environmentally related metal ions, compared to existinganodic stripping voltammetry (ASV) techniques. In addition,this sensor could be implemented using minimal reagents and working steps withexcellent reusability through mild regeneration procedure. It was expected that thiscost-effective electrochemical sensor might hold considerable potential in on-siteapplications of Hg2+detection.MicroRNA (miRNA) plays a crucial regulatory function in vivo and importantimplications on not only for cell growth, differentiation and apoptosis, but alsorelated closely with many diseases. The researchers found that miRNA are related tothe abnormal expression in leukemia, colorectal cancer, breast cancer andcardiovascular disorders diseases. Since miRNA is very short, less content andsimilarity characteristics than other sequences, analytical testing has somechallenges. The traditional method included the mark detecting hybridization,microarray technology, based RCA signal amplification detection techniques, andcationic polymers. In Chapter6of this paper, we reported double-invasive signalamplification based on highly selective sensitive miRNA detection methods andtechniques (Invader assay). Other researchers had invasive signal amplificationtechnology to detect miRNA, but their template can not achieve a good distinctionwith similar miRNA sequences. We designed the first heavy sucked miRNA invasiveprobe amplification cycle, and there is not formed immediately invade miRNA locioverlapped structure, we can learn the principles of notches ligated PCR, design ofmiRNA3’ end from the intrusion bit points nucleotides distance. When thethermophilic Taq polymerase was added, and the corresponding nucleosidetriphosphates, miRNA become fully matched primer extension to initiatepolymerization when the polymerization to the5’ end of the signal probe and probehybridizing region of the template (invasion locus), Taq polymerase5’-3’exonuclease activity began to play a role in the invasion site nucleotide probe signala (near the3’ end) of the disconnect between the nucleotide and the phosphatebackbone, the signal probe5’ tilt leave, but also because of another section of shortersequences hybridize with a template from the template and hybrid stability weakened,became the second heavy cycle amplification primers probes. The second cycle ofamplification similar to the first heavy weight cycling, also needs the role ofpolymerization to form intrusive structure, and further weaken the role ofnon-specific nicking, the second re-circulation of the probe signal is5’ fluorophorelabeled fluorescein, Taqman probes labeled quenching intermediate groups, butintrusive structure formation, the signal probe5’ cocked and with fluorescein modified left, middle, and signal probes labeled quencher separate fluorescencesignal enhancement and is proportional to the concentration of the target miRNAsequence. Experimental results show that the method can achieve double loopamplification step, high detection sensitivity, the lower limit of up to10fmol/L, inparticular, can distinguish with similar miRNA sequences, achieved a rare miRNAselective detection.