Studies Of Novel Fluorescence Biosensing Technology Based On Graphene Oxide

Rapid and sensitive detection of biomolecules has always been the researchinterest in biomedical and analytical chemistry. The rapid development of modernsociety imposes higher requirements on biosensing technology. Developing newanalytical techniques for solving more and more analytical problems and social crisishas proposed a great challenge for analysts. Fluorescent biosensing technology hasbeen widely applied in the detection of various biomolecules, including protein, smallmolecules, nucleic acids and enzyme activity, attributing to its analytical advantages ofhigh sensitivity and rapid response time. In order to improve the sensitivity, traditionalfluorescent biosensing technology usually requires multiple-fluorophore labeling anddifferent amplification techniques, which might add cost and complexity for the assay.In addition, traditional fluorescent biosensing technology suffers from a relativelyhigh background signal. Therefore, it is of great importance to develop new fluorescentbiosensing techniques so as to enhance the bioanalytical performance and reduce thecost.Due to its unique DNA absorbing ability and universal quenching properties forfluorophores, graphene oxide (GO) has been widely applied in biomolecular detection.GO-based fluorescent biosensors show several advantages compared with thetraditional ones, including improved assay sensitivity, low cost and enhanced signal tobackground ratio and so on. These advantages reveal that GO-based fluorescentbiosensors hold great potential in biomolecular detection. Most of these biosensorsrely on the fact that GO can adsorb the dye-labeled single-stranded DNA (ssDNA) andeffectively quench its fluorescence, the binding of analyte with dye-labeled ssDNAresults in the departure of the dye and GO, and the recovery of fluorescence which canbe used for target detection. However, these biosensors require fluorophore-labeledprobes and show relatively poor detection limits, which need further improvements.There has been report for biomolecular detection based on the combination of DNAinteracting dyes and GO, which shows an improved detection limit and it is expectedthat this kind of biosensor may find wide applications in biomolecular detection.Focusing on the above questions and making use of the achievements of previousresearchers, this thesis developed several novel fluorescent biosensing methods for thedetection of DNA enzyme activity, DNA and adenosine triphosphate (ATP) based on the unique DNA adsorption and fluorescence quenching property of GO. The detailsare described in following chapters.In Chapter2, we developed a novel label-free fluorescent strategy for thedetection of DNA3’ phosphatase and its inhibitors based on hairpin primer andpolymerase elongation. Only in the presence of DNA3’ phosphatase, DNA hairpinprimer with3’-phosphorylated end can be dephosphated, then polymerase elongationwas initiated to produce the double-stranded DNA (dsDNA). Because GO shows weakbinding affinity toward the SYBR Green I (SG) stained dsDNA that it cannoteffectively quench the fluorescence, thus one can obtain a strong fluorescence signal.Whereas, the hairpin primer would not be dephosphated in the absence of DNA3’phosphatase and its polymerase elongation would not occur. Adsorption of hairpinprimer by GO generates a rather weak background signal. This method can realize thesimultaneous detection of two kinds of DNA3’ phosphatase (T4PNKP and SAP) withachieved detection limits of0.07U/mL and0.003U/mL, respectively. In addition, thismethod is able to quantitatively evaluate the inhibition effect of the inhibitorcompounds and also applicable for complex biological sample.In Chapter3, a label-free sensitive fluorescent DNA biosensor was presentedbased on isothermal circular strand-displacement polymerization reaction (ICSDPR)combined with GO binding. This biosensor relies on the hybridization of target DNAwith hairpin probe as the prerequisite for ICSDPR and the preferential binding of GOto stained single stranded DNA (ssDNA) over double stranded one to improve thesignal to background ratio. By combining the properties of GO and SG with theICSDPR amplification, this fluorescent DNA biosensor displays a wide dynamic rangefrom0.01nM to10nM and a low detection limit of4pM. The proposed strategy issimple, cost-effective and sensitive, which might provide a promising method ofchoice for convenient DNA detection.In Chapter4, a simple, amplification-free and sensitive fluorescent biosensor forATP detection was developed based on the ATP-dependent enzymatic reaction(ATP-DER) and GO binding. In this assay, two half DNA probes anneal with adye-labeled template to form a DNA duplex substrate with a single-stranded nick. Theformed nick could be sealed by T4DNA ligase in the presence of ATP, and theresulting unnicked DNA duplex was resistant to thermal denaturation. Whereas, theligation reaction would not happen in the absence of ATP, and the nicked DNA duplexwas subject to thermal denaturation, which leads to the separation of two half DNAprobes and template. After addition of GO, the fluorescence of the nicked DNA duplex was greatly quenched, whereas efficient fluorescence quenching did not occur to theunnicked DNA duplex and high fluorescence intensity was obtained. The proposedstrategy was simple, amplification-free and showed high selectivity to ATP that coulddistinguish ATP from its analogues. The results revealed that the method allowedsensitive quantitative assay of ATP with a wide linear response range from0.5nM to100nM and a low detection limit of0.3nM.In Chapter5, we present a novel, label-free exonuclease Ⅲ-aided fluorescentassay strategy for ATP based on the ATP-dependent enzymatic reaction and GO. In thisassay, hairpin probe with5’ phosphorylated end anneals with complementary DNA toform a dsDNA complex with a single-stranded nick, which can be ligated by T4DNAligase in the presence of its cofactor ATP, resulting in a hairpin product with a stem oflong dsDNA. Because the five phosphorothioate nucleosides near the3’ end of hairpinprobe can hamper the Exo Ⅲ cleavage, thus the hairpin product after ligation couldkeep intact upon treatment with Exo Ⅲ. After addition of GO, a strong fluorescencesignal was observed. Whereas, the single-stranded nick cannot be ligated in theabsence of ATP, Exo Ⅲ cleaves the annealed dsDNA complex from the nick site anddigests the C-DNA, releasing the hairpin probe. GO selectively adsorb the hairpinprobe leading to efficient quenching of the fluorescence. This sensor displays animproved sensitivity and a wide linear range within the ATP concentration from1nMto200nM with a low detection limit of0.2nM. The proposed approach is simple,cost-effective and convenient, which might create a new methodology for developingsensitive ATP biosensor.In Chapter6, we developed a simple and novel fluorescent strategy for thedetection of endonuclease activity and its inhibitors based on hairpin probe and GO. Inthis assay, we designed a single dye-labeled hairpin probe that contains the recognitionsite for the endonuclease in the stem. Upon treatment with the target endonuclease,hairpin probe was digested into three pieces. One of the digested products, thedye-labeled short DNA would not be absored by GO, leads to a strong fluorescencesignal. In the absence of endonuclease, hairpin probe kept intact and was immediatelyabsorbed by GO, resulting in an effective fluorescence quenching. In addition, thismethod was applicable for inhibitor quantification. Given the simplicity, convenienceof this approach, the proposed method may provide an alternative approach for thestudy of the interaction between protein and DNA.