DNA Methylation Based On The Quenching Of Graphene And Its Small Molecular Analysis Method

With the improvement of medical technology, the mortality of the malignant diseases such as cancer has decreased, gradually, and cancer is no longer an incurable disease. The early diagnosis and treatment of various diseases is the key to improve the survival of patients with disease. Based on the research of the relations between genes and diseases related to genes such as cancer, it is of great importance to achieve early diagnosis and treatment of various diseases from the analysis of molecular level. Fluorescence spectrometry, which shows the advantages of high sensitivity, good selectivity, and simple operation, has been a crucial method for analysis. The development of fluorescent material, such as graphene and graphene quantum dots, makes great contributions to improving the detection and application of fluorescence sensor. The aim of thesis is to develop a fluorescence approach used for detection of methylated DNA and biological molecules based on the fluorescence properties of graphene materials in early diagnosis and treatment of diseases providing a quick, sensitive and accurate method. It has a high potential application in molecular diagnostics of disease in clinical environment. The main results are as follows:1. We report a new strategy for DNA transmethylase based on the fluorescence quenching of GO. The method is developed by designing a single-stranded probe that carries both a binding region responsible for facilitating the interaction with GO and a sensing region for specifically recognizing the target (T1) and hybridizing with it to form a P1/T1 duplex. The duplex is released from the GO surface through cleavage of Hpall, resulting in the recovery of fluorescence intensity of the fluorophore. However, this cleavage of HpaⅡ is blocked after the duplex was methylated by M.SssI, and therefore no recovered fluorescence signal can be detected. The magnitude of the recovered fluorescence signal is related to the MTase activity, establishing the basis of MTase activity assay. This assay can determine as low as ～(0.03 ± 0.01) U/mL (at a signal/noise of 3) of M.SssI with a linear range of 0.1 to 100 U/mL and has an ability to recognize the M.SssI from other MTases. The advantage of this assay is that it is capable of avoiding false signals arising due to the nonspecific adsorption of interferants, applying in rapid evaluation and screening of the inhibitors of MTase, and thus has a potential application in discovery of new anticancer drugs.2. This chapter reports a sensitive, and selective DNA demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with site-specific cleavage of HpaⅡ endonuclease for improving selectivity. This assay can determine as low as～(0.05±0.01) ng/mL (at a S/N of 3) of MBD2 with a linear range of 0.2 to 300 ng/mL and has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. It is sensitive and selective to detect the MBD2 activity containing complex component. Therefore, it can be expected to be used for the cancer risk assessment through monitoring DNA demethylase activity, and provide comprehensive and dependable information for the early detection of methylated DNA-related cancer.3. The boron-doped graphene quantum dots (BGQDs) are made by electrochemical method and applied in detection of hematin. Hematin-induced fluorescence quenching of BGQDs allows determination of hematin concentration in human erythrocytes directly. The method is sensitive and selective, in especial, the detection need not separate hematin from hemoglobin before doing the assay. There is a good linear relationship between AF and hematin concentration, ranging from 0.01 to 0.92 μM with the LOD being～(0.005±0.001) μM at an S/N of 3. This new method has potential for applications in clinical examinations and disease diagnoses. For example, the determination of the hematin level in two kind of red blood cell samples, healthy human erythrocytes and sickle cell erythrocytes, gives the average concentrations of hematin to be ～(23.1 ± 4.9) μM for healthy red cell cytosols and ～(52.5 ± 9.5) μM (average of two samples) for sickle red cell cytosols. We also discuss the mechanism of hematin-induced fluorescence quenching of BGQDs by coupling electrochemical method with DFT-based calculation and illustrate the feasibility of this method, theoretically. This new method is sensitive, label free, simple, and inexpensive and many tedious procedures related to sample separation and preparation can be omitted, implying this method has potential for applications in clinical examinations and disease diagnoses.