Sensitive New Nucleic Acid Fluorescent Probes Based On Enyme Signal Amplication Strategy

The biosensor is an emerging disciplinea originating from the interplay ofnumerous subjects. With the pushing of biological science, information science andmaterials science development, biosensor technology developed rapidly. Due to manyadvantages such as good selectivity, high sensitivity, simple operation, low cost andcontinuous on-line detection in complex system, biosensor has become essentialdetection methods and monitoring methods for the development of biotechnology.Currently, biosensors have been widely used in clinical diagnosis, research in the fieldof industrial control, food and pharmaceutical analysis, environmental protection andbiotechnology, and so on. With the continuous development of the analytical science,the ability of detection for various biomolecules in different conditions is increasinglyrequired. The development of the better performance of the biosensor is of paramountimportance for biomedical research and clinical diagnosis. Sensitivity and selectivityare important parameters of performance evaluation of sensor. The discovery of theperylene diimide derivative and functional nucleic acidsoffer totally new designideals and platform for the biosensor system targeting on all kinds of compounds ofinterest.In view of the considerations above and many relevant documents, in this masterthesis, enhance the sensitivity of the biosensor is our main purpose. We combinefunctional nucleic acid and perylene diimide derivativeto build several newfluorescent sensors. The details are summarized as follows:(1) Study on the biosensors based on perylene diimide derivative (compound1).Strong electrostatic interactions between the cationic compound1and the polyanionicnucleic acid induced the aggregation of compound1and resulted in the fluorescencequenching. Thus, we use cationic aggregated perylene1as a broad-spectrum andlabel-free quencher which is able to efficiently quench a variety of anionicoligonucleotide-labeled fluorophores. In chapter2, by choosing nucleases as modelbiomolecules, such a broad-spectrum quencher was then employed to construct amultiplexed bioassay platform. The nucleases EcoRI and EcoRV were taken as themodel analyte employed for endonuclease activity detection. EcoRI and EcoRV caneffectively recognize specific sequences and cutthe restriction sites on dsDNA. In theabsence of nuclease, the fluorescence of the DNA-labeled fluorophore will be efficiently quenched. Upon addition of the nuclease, the oligonucleotide probe will becleaved on the cleavage site, releasing a short fluorophore-conjugated ssDNA. Sincethe short fluorophore-linked oligonucleotide only contains5bases, which could binda few molecules of derivative1, therefore, compound1will exhibit weak quenchingeffect on the fluorophore’s fluorescence. The fluorescence intensity will graduallyincrease with the addition of increasing concentrations of nuclease. Under optimalconditions, the sensor exhibits high sensitivity to nuclease with a detection of limit of0.03U/mL for EcoRV, and0.05U/mL for EcoRI. Using the compound1as superfluorescence quencher and the Exo III as an amplifying biocatalyst, we develop afacile, sensitive, cost-effective method for multiplexed DNA detection in chapter3. Inthe absence of target DNA, Exo III is unable to catalyze the removal of bases from theprobe DNA. In the presence of target DNA, Exo III can catalyze the stepwise removalof mononucleotides of probe DNA from the blunt3′termini, which resulting in thereleasing of the target and fluorophore. So that the corresponding fluorophorefluorescence recovery. The released target DNA can hybridize with another probeDNA and then initiate a next round of cleavage. This method can be used to detectmultiple DNA target. Under the optimized experimental conditions, the proposedbiosensor exhibits high sensitivity, and a detection limit of20pM could be achievedfor target DNA.(2) Study on DLISA based biosensor. In chapter4, reported a DLISA approachusing DNAzyme for ELISA detection. A protease replaced by DNAzyme, whichresulting in an alternative to the classical ELISA immunoassay that is more stable byits protein enzyme-free design. It is comprised of three key components: anantibody-modified96-well plate, a ZnS-conjugated secondary antibody and a CAMBsystem. We choose human IgG as model biomolecules. In the presence of the targetantigen, the ZnS-conjugated secondary antibody is captured on the antibody-modifiedwell of the96-well plate by the formation of a sandwich immunocomplex. Subsequentaddition of Ag+triggers the CX reaction to release Zn2+from the ZnS NCs. Each ionicNC can release thousands of Zn2+into solution by the CX reaction. Since oneDNAzyme can catalyze the cleavage of several substrates, the CAMB system affordsan amplified signal through cycling and regenerating the Zn2+-dependent DNAzymeto realize multiple enzymatic turnovers. By multiple signal amplification inexperiments, the sensor to obtain a high sensitivity with the detection limit of2fg/mL. It also exhibited high selectivity，which make it valuable for the detection oftarget biomolecule in complex biological samples. We also examine the versatility of the sensor, together with the detection of rabbit IgG, mouse IgG and human IgG, theresults showed that the sensor has a good response performance of rabbit IgG, mouseIgG. This study demonstrates the potential utility of such enzyme-free,triple-amplified DLISA strategy in multiplex biological analysis.