Studies Of Ultrasensitive Fluorescent Biosensing System Based On DNAzyme And Graphene Oxide

Biosensor is an emerging and active field originating from the interplay ofnumerous subjects, such as biology, physics, mathematics, chemistry and computertechnology. Recent years, as the new detection technologies constantly spring up andget perfected both in the research and practical application, the sensitivity and thespecificity of the biosensor have been largely improved. However, with the researchdeveloping, the development of the sensitive, selective and inexpensive biosensorsare of paramount importance for biomedical research and clinical diagnosis. Twodifferent strategies have been widely employed to improve the sensitivity of a sensor:lower the detection background and signal amplified detection.In view of the considerations above and many relevant documents, we mainlyutilize the super fluorescence quencher and signal amplified strategy to improve thedetection sensitivity of the biosensor. The major contents are as follows:（1） Study on the biosensors based on DNAzyme. DNAzymes （also calleddeoxyribozymes or DNA enzymes） are DNA sequences that can catalyze chemicalreactions, such as cleaving ribonucleic acid targets. Based on the remarkabledifference in affinity of GO with ssDNA containing different number of bases inlength, we constructed a graphene–DNAzyme based sensing system for amplifiedfluorescence “turn-on” detection of Pb²⁺in chapter2. The DNAzyme-substratehybrid containing a large ssDNA loop make the fluorophore close to the GO surfaceto the greatest extent to afford a high quenching efficiency and a low backgroundfluorescence. Upon the addition of Pb²⁺, the DNAzyme was activated and cleaved thesubstrate strand, releasing a short FAM-linked oligonuleotide fragment, a relatedlong oligonuleotide fragment and the DNAzyme strand. The DNAzyme strand canhybridize with another substrate strand and then induce the second cycle of cleavageby binding Pb²⁺. The introduction of GO into the sensing solution will result in weakquenching of the fluorescence of FAM due to the weak affinity of the shortFAM-linked oligonuleotide fragment to GO, and the fluorescence intensity shouldgradually increase with increasing Pb²⁺concentration added. Our proposed biosensorexhibits a high sensitivity towards target with a detection limit of300pM for Pb²⁺.In chapter3, we employ cationic perylene derivative as a superquencher toconstruct DNAzyme-based fluorescence catalytic biosensors for the detection of Pb²⁺ and L-histidine. The strong affinity of perylene derivative with DNA drives it to forma complex with the DNAzyme-substrate hybrid, and efficiently quench thefluorescence of the FAM moiety. The introduction of Pb²⁺, however, will activateDNAzyme to catalyze the cleavage of the substrate in the RNA site, releasing a shortFAM-linked oligonuleotide fragment which show weak affinity with perylenederivative, and the introduction of perylene derivative into the system will showweak quenching effect on the FAM’s fluorescence. These results togetherdemonstrate that our design provides a convenient and universal platform foramplified detection of a wide range of targets.The molecular beacon has the properties of low background and high sensitivity,and the huge difference with the melting temperature of other double-strand DNA.By utilizing the molecular beacons as the substrate sequence, a new kind offluorescent catalytic and molecular beacon has been developed in chapter4.Moreover, this design is willing to be applied in the fluorescence-based signalamplification for the detection of target DNA. A dynamic range from0.5nM to300nM for target DNA was achieved and the detection limit was200pM.In chapter5, taking advantage of a topological effect of DNAzyme, we designeda new and general amplified sensing platform for nucleic acids and Dam MTaseactivity detection. Because the8-17DNAzyme is caged by partially hybridizing toform a hairpin structure, and is inactive to MB substrate, which provides a lowbackground for the sensing system. In the presence of the target, the hairpin structureis opened, and the8-17DNAzyme is liberated from the caged structure with itscatalytic activity being restored. The activated8-17DNAzyme can hybridize with theMB substrate to form the CAMB system and catalyze the cleavage of the MBsubstrate in the presence of cofactor Zn²⁺. After cleavage, the quenched MBfluorophore/quencher pair was separated from each other, resulting in an obviousincrease of fluorescent signal and a free DNAzyme strand. Eventually, eachtarget-induced activated8-17DNAzyme can undergo many cycles to trigger thecleavage of many MB substrates, achieving an amplified detection signal for thetarget. Furthermore, we constructed an amplified sensing platform for proteindetection based on the terminal protection of small-molecule-linked DNA. In theabsence of SA, the DNA probe was hydrolyzed successively into mononucleotides byExo I and cannot catalyze the cleavage of the MB substrate, therefore, providing azero-background for the sensing system. In the presence of SA, the probe is protectedfrom the degradation by Exo I. Consequently, the probe containing the8-17 DNAzyme sequence can catalyze the cleavage of the hairpin-structured MB substrateto induce a significant fluorescence enhancement.（2） Using the GO as super fluorescence quencher and the Exo III as anamplifying biocatalyst, we develop a facile, sensitive, cost-effective method for DNAdetection in chapter6. In the absence of target DNA, Exo III is unable to catalyze theremoval of bases from the probe DNA. Upon the addition of GO, the fluorescence ofFAM was significantly quenched because of the strong binding between ssDNA andGO. In the presence of target DNA, Exo III can catalyze the stepwise removal ofmononucleotides of probe DNA from the blunt3′termini, which resulting in thereleasing of the target and fluorophore. The released target DNA can hybridize withanother probe DNA and then initiate a next round of cleavage. The introduction ofGO into the sensing solution will induce weak quenching of the fluorophore becauseof the weak affinity of the fluorophore and GO, and the fluorescence intensitygradually increases with increasing target concentration. The proposed biosensorexhibits high sensitivity, and a detection limit of20pM could be achieved for targetDNA.（3） In chapter7, we report a simple, rapid and label-free approach forfluorescent “turn-on” detection of Pb²⁺by using a water-soluble cationic perylenederivative （compound1） as the fluorescence reporter and the G-rich DNA probe forthe specific binding of Pb²⁺. Strong electrostatic interactions between the cationiccompound1and the polyanionic nucleic acid induced the aggregation of compound1and resulted in the fluorescence quenching. Upon the addition of Pb²⁺, theconformation of DNA probe changed from a random-coil to a Pb²⁺-stabilizedG-quadruplex structure. This conformational change weakened the interactionbetween probe DNA and compound1. As a result, compound1is present in both thefree monomeric and the aggregated form in aqueous solution. Owing to the existenceof the free dye monomer, a fluorescence enhancement was observed. A dynamicrange from10nM to10μM for Pb²⁺was achieved and the detection limit was4nM.