Studies Of Novel Optical Biosensing Technology Based On Enzyme Signal Amplification And The Nanomaterials

The study of drug molecules and small biomolecules has great significance fordrug screening, clinical diagnosis and therapy. In order to solve more and moreanalytical problems and social crisis, the development of novel, simple andcost-effective biosensing strategies with high sensitivity and selectivity is still a greatchallenge to analysts. Therefore, a series of novel optical detection technologies basedon enzyme (G-quadruplex DNAzyme, DNA polymerase and nickase) signalamplification and the nanomaterials were developed for drug molecules and smallbiomolecules detection, respectively. Compared with the traditional methods, thedeveloped detection methods are sensitive, selective and fast, and provide referencesfor the rapid development of biosensing technology.(1) Coralyne is a polydeoxyadenosine [poly(dA)]-binding drug with noticeableantitumor activity. In Chapter2, we developed a simple colorimetric strategy throughthe mechanism of target induced split G-quadruplex formation for detecting coralyne.The designed two DNA oligonucleotides both containing split G-quadruplex sequenceand adenine-rich sequence are used in our strategy. In the presence of coralyne, theadenine-rich sequences of two oligonucleotides are drawn in close proximity to form astable duplex, resulting in the formation of split G-quadruplex DNAzymes thatcatalyzes the generation of a colored product. The DNAzyme-based colorimetric assayfor coralyne is simple, cost-effective, visible and highly sensitive with a low detectionlimit of16nM. The developed sensing platform holds great potential for applicationsin drug screening.(2) Existing strategies for detecting nicotinamide adenine dinucleotide (NAD+) orother cofactors are commonly cumbersome and moderate sensitive. In Chapter3, wereport a novel DNAzyme-based visual assay strategy for NAD+based onligase-mediated inhibition of the strand displacement amplification (SDA). In thepresence of NAD+, the two primer DNAs are ligated to an integrated primer which caninhibit SDA by the integrated primer’s3’ mismatched bases. In the absence of NAD+,with the aid of polymerase and nickase, the extension process can proceed to produce agreat amount of G-quadruplex DNAs via SDA, the formed G-quadruplex DNAzymecan quantitatively catalyze the formation of a colored product. Therefore, thequantitative analysis for NAD+can be achieved visually with high sensitivity. The developed strategy provides a simple colorimetric approach with high selectivityagainst most interferences and a detection limit as low as50pM. It also provides auniversal platform for investigating cofactors or other related small molecules as wellas quantifying the activity of DNA ligases.(3) MicroRNAs (miRNAs) play vital roles in physiologic and pathologicprocesses and are significant biomarkers for disease diagnostics and therapeutics. InChapter4, we develop a new strategy for simple and sensitive detection of miRNA bycombining WS2nanosheet based fluorescence quenching with duplex-specific nucleasesignal amplification (DSNSA). Upon the addition of target miRNA, the ssDNA probehybridizes to the target miRNA to form DNA/RNA heteroduplex, the DNA sequence ofthe DNA/RNA can be cleaved by duplex-specific nuclease (DSN) and the targetmiRNA can be released to initiate a next round of hybridization, cleavage andreleasing. This cyclic reaction leads to a great amplification of FAM-linkedoligonucleotide fragments to show strong fluorescence signal. In contrast, in theabsence of target miRNA, the DSNSA reaction would not take place, the ssDNAprobes remain intact and their fluorescence is almost entirely quenched due to theirstrong affinity to WS2nanosheets.This assay exhibits highly selectivity with adetection limit of300fM and even discriminates single-base difference between themiRNA family members. The result indicates that this simple and cost-effectivestrategy holds great potential application in biomedical research and clinicaldiagnostics.