Constructions Of Nucleic Acids Probes Based On Graphene Oxide And Their Biosensing Applications

Along with the progress and development of society and people's living conditions, people are increasingly concerning about their own health and hoping to have a higher quality of life and longer lifetime. Therefore, people are eager to understand the internal biochemical information of their own. Under this circumstance, biosensor is developed as a device for the detection of analytes that combines a biological component with a physical detector component. A biosensor contains at least three components:target recognition element, signal transduction element and detector element. The target recognition element can be any chemical or biological entity such as small organic molecules, peptides, proteins, nucleic acids, carbohydrates, or even whole cells. When the DNA molecule is recognized as the recognition element, the biosensor is called DNA biosensor. Single-stranded DNAs or RNAs can bind to their complementary strands with high specificity and are useful for nucleic acid detection. With the development of aptamer, DNA biosensor can be used to detect a diverse range of analytes beyond DNA or RNA with high affinity and specificity.Recently, graphene oxide (GO) has been introduced to construct DNA biosensors based on the principle of fluorescence resonance energy transfer (FRET). GO is an excellent energy acceptor in FRET and makes the fluorescent detection as a promising application of GO in sensing technology.Based on the excellent quenching ability of GO, we herein designed fast, low-cost, high accuracy, high selectivity and high sensitivity of DNA biosensors for biological small molecule ATP and different types of proteins that interact with DNA in different ways. Besides, we investigated the applicability of these analytical techniques to the analysis of real samples. The research work of this dissertation is summarized as follows:1. Detection of small biological molecule ATP. When aptamer-target interactions are involved in the design of molecular beacon (MB) which is mainly used for DNA detection, it is called molecular aptamer beacon (MAB). Although traditional MAB holds the advantages of both MB and aptamer, it has to be labeled with both the fluorophore and the quencher, which lead to complications and high-cost. Moreover, it has strong background because of the low FRET efficiency between the conventional fluorophore and quencher. Herein, a novel GO-based MAB was designed to overcome the shortcomings of the traditional MAB for ATP detection. The results show that ATP can be detected in a wide range of5-2500?M with a detection limit of2?M and good selectivity. Moreover, the GO-based MAB can be used to detect the extracted ATP from living cells.2. Detection of a protein which binds to DNA specifically. The specific binding reaction between aptamer and protein can be used for the detection of DNA-specific binding protein. Herein, a highly sensitive and selective fluorescent aptasensor for mucin1(MUC1) detection is constructed, utilizing GO as the quencher that quenches the fluorescence of single-stranded dye-labeled MUC1specific aptamer. Using this method, MUC1can be detected in a wide range of0.04-10?M with a detection limit of28nM and good selectivity. Besides, this method does not need the assistance of the antibody and enzyme labeling thus making it simple and low-cost. Moreover, it has also been verified for real sample application by testing2%serum containing buffer solution spiked with a series of concentrations of MUC1.3. Detection of a protein of the enzymes which having a hydrolysis role of DNA. The detection of enzyme activity is of great importance in biomedical analysis. Herein, an ultra-high sensitive and selective fluorescent sensing platform for micrococcal nuclease (MNase) is developed based on MNase-induced DNA strand scission and the difference in affinity of graphene oxide (GO) for single-stranded DNA containing different numbers of bases in length. Using this method. MNase can be detected in a range of8×10-5-1.6×10-3units/mL with a detection limit of2.7×10-5units/mL and good selectivity. The detection limit is of two orders of magnitude lower than those reported fluorescence MNase assay. Besides, this GO-based biosensor can be used to detect MNase secreted by Staphylococcus aureus (S. aureus). Moreover, this method can be easily extended to the detection of other nuclease, by simply changing the working buffer.4. Detection of a protein which can not interact with DNA directly. Using oligonucleotide to build biosensors has unique advantages because oligonucleotide molecule is small, simple and reproducibly synthetic, thermally stable, reusable, and long-term stable. However, some proteins can not interact with oligonucleotide directly. In order to take full advantage of DNA as a platform to build biosensors, indirect method can be used for the detection of such class of proteins. Herein, based on terminal protection of small molecule-linked DNA by target proteins and GO- assisted DNA assay strategy, we have developed a novel fluorescent method for the detection of target protein folate receptor (FR). Using this method, FR can be detected in a range of1-800ng/mL with a detection limit of0.77ng/mL and good selectivity. Besides, this method can also be used to detect FR in2%serum solution. Moreover, this GO-based biosensor can be used to detect other proteins by simply changing the small molecule at the terminal of DNA.