| DNA-enzyme conjugates both have the structure-directing properties of DNAand various functionalities of enzymes. Such DNA-enzyme conjugates, with theircombined properties, would find a broad range of applications, including biomedicaldiagnostics, molecular recognition, nanotechnologies, enzyme cascade reactions. Twomethods have been developed to prepare DNA-enzyme conjugates, covalent approachand non-covalent approach. For most approaches, the conjugation between a largeDNA and a large enzyme molecule often results in a moderate yield. As a result,considerable amounts of unreacted DNAs and enzymes are still present in the reactionmixture, which may cause high blank signal and low sensitivity.To avoid interference from unreacted DNAs and enzymes, chromatography andcommercial separation columns are used to purify DNA-enzyme conjugates.However, it is difficult to separate the conjugates from DNAs and enzymes of similarmolecular weight. Other properties of the enzymes, such as hydrophobicity andisoelectric point may also need to be taken into account to optimize the purificationprocedures. Hence, the aim of this dissertation is to design a simple and efficientmethod to purify DNA-enzyme conjugates and apply the purified conjugates fordeveloping highly sensitive and selective detection methods for biomolecules.Firstly, our purification method is based on the different association constantsbetween biotin-streptavidin and desthiobiotin-streptavidin. The much strongerbinding affinity of streptavidin toward biotin over desthiobiotin is the workingprinciple of displacement reaction. The applications of purified DNA-invertaseconjugates in a structure-switching assay for cocaine and a competitive hybridizationassay for DNA were demonstrated, both experiments showed enhanced analyticalperformance compared with the same assays using unpurified DNA-invertaseconjugates. Limit of detection for cocaine was found to be1.8μM by using purifiedconjugates but was4.2μMwhen using unpurified conjugates. To further demonstratethe advantage of using purified conjugates, the purified DNA-invertase conjugates were applied in a competitive DNA hybridization assay. For purified conjugates, avery low blank signal (26mg/dL) and a detection limit of2.1nM were found. Incontrast, if unpurified conjugates were used for the same assays, the blank signal wasfound about8times as high as that when using purified conjugates (213mg/dL) andthe detection limit is65.2nM DNA.Secondly, a new colorimetric method for caspase3activity assay was developedby taking advantages of the catalytic properties of HRP-mimicking DNAzymes andthe specific cleavage of DEVD-peptides by active caspase3. Under optimalconditions, the detection limit of caspase3was0.89nM. Thrombin and trypsin wereused as control proteases, only caspase3could induce a significant absorption change,suggesting the high selectivity toward caspase3. The proposed method was alsosuccessfully applied for the detection of caspase3in apoptosis Hela cell lysates.Finally, the catalytic activity of8-17DNAzyme was investigated by extendingits sequence in this dissertation. The activity of8-17DNAzyme was significantlyinhibited in the “close†state. In the presence of a target substance, the activity of8-17DNAzyme was recovered. The approach was successfully utilized for quantitativedetection of adenosine and IFN-γ. |
