Development Of New Biosensing Method Based On Gold Nanoparticles And Nucleic Acid

Nano-materials exhibit a series of unique properties in mechanical, optical,electrical, magnetic and catalytic, due to their quantum size effect, surface effect,small size effect and macroscopic quantum tunneling effect. Therefore, it has beenincreasingly widely used in many areas. Of all the nano-materials, gold nano-materialis among one of the most extensively applied nano-materials in various fields. Inrecent years, the gold nano-material applications has become a hot research and focusin biosensor. And biosensor has been widely used in medical diagnosis, clinical testing,bio-chemical, food industry and environmental testing and other aspect, goldnano-material provides a new idea for biosensor research and application because of itsstrong adsorption capacity, good directional ability and biological compatibility.As a biosensing tool, DNA probe can convert relevant information of life intoeasily detected signal (fluorescence, Raman, chemiluminescence and electrochemicalfor example), skilfully using some specific chemical properties of biological molecules,such as nucleic acid hybridization, the specific bingding of protein, enzymebiochemical function and structure-switching, and combining with signal transductionmechanism of molecular level. Now, because of its high specificity, low backgroundsignal and easy to realize detection of living cells, DNA probe has become a hotreseach biosensing. In this paper, we have developed a series of novel, fast,easy-operation, low-cost optical and electrochemical analysis method for selective andsensitive detection of metal ions, DNA, proteins and cancer cells, utilizing Au NPs andDNA probe, combined with the developed methods and techniques in our group. Thedetailed contents are described as follows:(1) In chapter2, we constructed a simple, rapid, label-free fluorescencebiosensing method for sensitive deteciton of silver ions based on structure-switchingDNA. In this method, we designed a silver ion specific DNA, which is a C-richoligonucleotide DNA. In the presence of Ag+, silver-mediate base pairs (C-Ag+-C) ofsilver ion specific DNA could be formed between cytosine residues from two Ag+-binding sequences to give rise to a hairpin structure. In the hairpin structure, the SybrGreen Ⅰ binds to the silver ion specific DNA hairpin to achieve the high Sybr GreenⅠ emission intensity. In the absence of Ag+, the silver ion specific DNA exists as arandom coil which showed a very weak fluorescence. The whole detection process was basically done in a single step, and did not require any tags, which was not onlyprovides a simple, fast, label-free method for the detection of silver ions, but alsoprovides a new way of thinking for the detection of metal.(2) In chapter3, we exploited a label-free, homogeneous biosensing platformbased on plasmonic coupling and surface-enhanced Raman scattering. This method iscarried out by unmodified gold nanoparticles for SERS detection: first, there aredifferences in adsorption properties of ssDNA and dsDNA on citrate-coated Au NPs.The ssDNA adsorbs efficiently on the Au NPs surface through strong electrostaticattraction while dsDNA does not, which results in ssDNA that can protect the Au NPsfrom salt-induced aggregation, but dsDNA cannot. Secondly, the aggregation ofmonodis-persed Au NPs into discrete clusters can give rise to the plasmonic couplingand the formation of many SERS ‘‘hot spots’’, resulting in the enormous enhancementof Raman scattering. We designed two ssDNA probes for this sensor platform indetection of DNA and Hg2+respectively: one was perfected complementary to a targetDNA, and the other one was a specific DNA to Hg2+. In the presence of target, thetarget was hybridized or binded with its corresponding ssDNA probe. After theaddition of unmodified Au NPs, Raman reporter molecules and NaCl, the aggregationof Au NPs occurs due to lack of the protection of ssDNA, resulting in a strong SERSsignal. On the contrary, in the absence of target, the ssDNA adsorbs on the Au NPssurface and protects Au NPs from salt-induced aggregation, leading to a weak SERSsignal. This assay possesses the superior signal-to-background ratio as high as~30andexcellent selectivity, and not only simple in design but also quick, easy in operation.Factually, this method can in principle be extended to detect various analytes, such asother metal ions, proteins and small molecules by using the oligonucleotides that canselectively bind the analytes.(3) In order to further demonstrate the generality of the strategy in chapter3, weused the same principle to develop a target-controlled plasmonic coupling andSERS-based biosensing technique for the detection of Melamine using unmodifiedAuNPs in chapter4. In this strategy, we designed a poly-T oligonucleotide DNA,which can combine with melamine through hydrogen bonds to form a T-melamine-Tstructure. First, AuNPs are decorated through mixed self-assembly with polythymineand Raman-active dye via electrostatic attraction. By adsorbing polythymine onto thesurfaces of AuNPs, the negative charges of polythymine can maintain AuNPswell-dispersed in the reaction mixture and protect AuNPs from salt-inducedaggregation owing to the repulsion force of the neighboring nanoparticles. Then, the present melamine decreases the negative charges of the surface of AuNPs by formingtriple H-bonds with polythymine in aqueous medium, thus leading to thedestabilization of AuNPs and the aggregation of AuNPs with the addition of NaCl. Asa result, strong interparticle plasmonic coupling between AuNPs can be induced withremarkable enhancement of the Raman signal, due to dramatically enhancedelectromagnetic fields near the particle surface. Due to the high efficiency oftarget-controlled interparticle plasmonic coupling and SERS enhancement, thisnanosensor exhibits high sensitivity and selectivity in rapid melamine assay, with aquite low detection limit of8nM for real liquid milk samples.(4) In chapter5, we presented a novel electrochemical platform for sensitivedetection of cancer cells by using Aptamer-aided target capturing with biocatalyticmetal deposition. In this chapter, we chose Ramos cell as a model case, and designdtwo Aptamer probes with different sequences and binding sites of the same cancer cell:Aptamer probe1is thiolated at the5’ end and via covalent binding, its thiolated endcan be immobilized onto the surface of the gold electrode. Aptamer2is a probebiotinylated at its5’ end for the silver deposition reaction on the electrode surface bybinding with SA-ALP. First, the thiolated probe1immobilized on the surface ofworking electrode, which is also the Aptamer for the target cell, recognises andspecifically captures the target cell close to the electrode. In the presence of probe2, itfurther specifically binds with the captured cancer cell, forming “Aptamer-Ramoscell-Aptamer” composites. As probe2is biotinylated, it can conjugate with theSA–ALP through the biotin–streptavidin interaction. Thus, it allows the nonreductivesubstrate of alkaline phosphatase, ascorbic acid2-phosphate, to be converted intoreducing agent ascorbic acid on the electrode surface. Hence, the silver ions arereduced and deposited on the electrode surface. Furthermore, as the Aptamer sensorutilizes the enzymatic silver deposition procedure for electrochemical quantifcation ofthe target cell, it permits the accumulation of the enzymatic product at the electrodesurface for a highly sensitive LSV readout.The developed strategy was demonstrated to display high selectivity indiscriminating Ramos cells, detection limit as low as10cells, and a wide linearresponse range10to106cells with desirable reproducibility. The technique platformwas proved to be cost-efficient with excellent compatibility to miniaturizationtechnologies. These properties support its potential in clinical applications. Moreover,multiplex detection of multiple cells can be implemented in densely packed arrayformat with specific Aptamers selected for each kind of cell. In view of these advantages, this new electrochemical cell detection strategy is expected to provide anintrinsically specific and sensitive platform for cancer cell assay and associatedstudies.(5) In chapter6, we developed a novel electrochemical immunoassay for thedetection of human IgG by using AuNPs and telomerase extension reaction as dualsignal amplification and coupling biocatalytic silver deposition. In this assay, weselected human IgG as a target protein, and introduced a Ab-Au NPs-P1nanocomplex,which were Au NPs modified with telomerase primer DNA probe1and goatanti-human IgG (Ab). Firstly, goat anti-human IgG (Ab) is immobilized on goldelectrode through cysteamine and glutaraldehyde reaction. After electrode surfaceblocked by BSA, analyte human IgG antigen is bonded with Ab specifically. ThenAb-AuNPs-P1nanocomplex is used as a secondary antibody to combine human IgGantigen to form a sandwich structure. In the presence of telomerase, telomeraseextension reaction is initiated to add TTAGGG tandem repeat unites to the3’-end ofthe primer by adding nucleotide mixture dNTPs. DNA probe P2is designed forhybridization with telomerase extension product. Then streptavidin labeled alkalinephosphatase (SA-ALP) is employed to connect ALP onto electrode surface via specificbinding between biotin and streptavin. After washing steps, the modified goldelectrode is incubated with ascorbic acid2-phosphate (AA-P) and silver ions. The ALPconverts AA-P to ascorbic acid, a reducing reagent which reduces silver ions to form ametallic silver layer on the electrode surface. The amount of deposited silver iscorrelated with analyte human IgG concentration and can be determined by linearsweep voltammetry (LSV). The deposited silver layer can only be obtained in thepresence of human IgG.This assay possesses the superior signal-to-background ratio as high as~35, andthe detection limited was as low as0.02μg/mL. Hence, this strategy held the potentialto be extended to a common electrochemical immunoassay platform for differentproteins assay. The real human serum analysis results demonstrated the developedapproach could be used for quantitative analysis of practical and clinical samples.