Development Of DNA Biosensing Technologies With Emerging Nanomaterials

Nanomaterials are playing an increasingly important role in the development ofbiosensors. The sensitivity and performance of biosensors is being improved by usingnanomaterials for their construction. The use of these nanomaterials has allowed theintroduction of many new signal transduction technologies in biosensors. The combination ofnanomaterials and biological sensing elements to selectively recognize chemical or biologicalmolecules has resulted in the development of novel nanobiosensors. Nanobiosensors offerseveral important advantages over conventional biological procedures, and could have asignificant impact on humankind. Hence, the momentum toward building miniaturized,reliable, sensitive, and selective sensing instruments has focused on combining nanomaterialswith biomolecules for detection of a wide range of analyses. In this thesis, we describeseveral emerging nanomaterials that apply for DNA biosensing technologies.Firstly, we used enzyme-catalyzed assembly of gold nanoparticles for visualizedscreening of DNA base excision repair as reported in Chapter2. Activity screening of DNAbase excision repair (BER) enzymes is a crucial step for understanding numerousfundamental biochemical processes. A novel label-free homogeneous technique is developedfor visualized uracil-DNA glycosylase (UDG) activity assay using gold nanoparticles(AuNPs). This strategy relies on the enzyme-catalyzed assembly of AuNPs decorated withDNA probes. In the presence of endonuclease IV (an enzyme which can further hydrolyze theproducts from UDG-catalyzed reaction), the substrate DNA selectively interacts with UDGfollowed by the efficient release of single-strand probe. The released single-strand probe thenmakes the network-like assembly of decorated AuNPs to provide a visible signal for UDGactivity. This strategy that can be performed in a label-free homogeneous assay formatimproved the duration, the simplicity and the throughput of UDG activity screening. Theresults provided in the present study revealed that this strategy could have great potential as arobust, convenient and visualized platform for activity screening of UDG with highselectivity and desirable sensitivity. To increase the accuracy and diversity of UDG activitydetection, we describe another method that is a low-cost, high-speed, sensitivity, simple DNAbiosensor to detect the activity of UDG. The principle of the method is based on the fact thatfluorescence of SYBR Green I will be strengthened hugely after combining withdouble-stranded DNA (dsDNA) with and without UDG. In the presence of UDG, the uracilon dsDNA will be removed and broken though structure of dsDNA; leading to decrease in thefluorescent intensity of system. The results of experiment showed that the fluorescence of SYBR Green I decreases with increase the concentration of UDG. In addition, someconditions and working curve of UDG are established, with0.005U/mL of the detection limit.Unlike traditional gel electrophoresis and radioactive, this fluorescence assay easily providedus with the ability to detect the activity of UDG in homogeneous solutions. This method wassimple and non-radioactive, yet remained as efficient as gel electrophoresis. We used this newmethod to screen suitable inhibitor drugs of UDG.Secondly, we report a new strategy to study O6-methylguanine DNA methyltransferase(MGMT) activity using DNA-templated silver nanoclusters as a signal indicator enzymesactivity in Chapter3. A hairpin DNA probe is prepared with a nanocluster nucleationsequence and a fluorescence enhancement of silver nanoclusters using guanine-rich DNAsequences linked at the5’ and3’ terminus of the probe. The first, MGMT could excise themethyl group at O6position of guanine in the hairpin probe; subsequently Pvu II digested atguanine position of5’-G-A-G-C-T-G-3’ recognition sequence in the hairpin probe, resultingin fluorescence intensity change. Quite a wide dynamic range from0.001to1.0μg/mL wasachieved for MGMT assay and the detection limit was estimated to be8.0ng/mL. The resultsindicated that this strategy offers a simple, cost-effective, highly sensitive and selectivehomogeneous detection platform for MGMT activity assay related biochemical studies.Then finally, A DNA biosensor has been used for the determination of melamine inChapter4. The method is base on non-covalent assembly of aptamer (T55) on graphenewhich is induced by π-π stacking of DNA bases on graphene. The mixture of T55and GO,shows weak fluorescence owing to the strong adsorption of T55on GO surface via the π-πstacking attraction and super fluorescence quenching ability of GO, because of poly-Tnucleotide chain corresponding to a single stranded DNA that binding force of SG withsingle-stranded DNA is relatively weak. Upon the addition of melamine, competitive bindingof melamine and GO with T55causes the release of T55from GO surface, allowingfluorescence-signal enhancement. The enhancement factors increase linearly with melamineconcentrations over the range10to200nM. Beside desirable sensitivity, the developedstrategies also offer high selectivity, excellent reproducibility, low cost and simplifiedoperations, implying that these techniques may hold considerable potential for moleculardiagnostics and genomic researches.