Research On Novel Biosensing Technology Based On Carbon Nitride Nanosheets

The continuous development of science and technology prompts the development of life science,and the knowledge of life science is also deepening.Therefore,it is particularly important to rapidly,sensitively,accurately detect and analyze some biological molecules（such as enzymes,nucleic acids,etc.）that play key roles in living organisms.In recent years,as the core of nanotechnology,nanomaterials have been becoming the hot research topic because of its unique optical,electrical,catalytic performance.They were widely applied in various fields including optoelectronics,biological medicine,microelectronics,environment,energy and so on.As the combination product of nanomaterials with biosensors,nanobiosensors were integrated with multiple disciplines（such as chemistry,biology,physics and nanoscience,etc.）and their emergence highly facilitated the development and employment of biosensors in different fields.The development of novel detection principle and sensing mechanism of nanobiosensors,greatly improved the analytical performance of the biosensors and unquestionably broaden their application in the medical diagnosis and treatment,food analysis,environmental testing and other fields.Adenylate kinase（ADK）,an important phosphotransferase,is involved in the catalytic interconversion of the adenine nucleotides in variety of organisms,maintains the balance of adenine nucleotides,which can keep energy balance and promote high energy storage and utilization of adenine nucleotides.Single nucleotide polymorphism（SNP）refers to the polymorphism of DNA sequences caused by a single nucleotide mutation at the genome level.SNP is considered to be the most important form of variation in the human genome,and it is also the most common genetic variation in human genetic variation.SNP has a very important connection with the study of gene function,which can provide important basis for the diagnosis of disease and the research of individual drug response.In this thesis,two-dimensional carbon nanomaterials,graphitic carbon nitride nanosheets（CNNS）,was used to develop rapid and label-free biosensing strategies for detecting disease biomarker ADK and SNP.The specific contents of the study are presented as follows:In the second chapter,we developed a new CNNS-based biosensing platform for simple,sensitive and label-free detection of ADK.The strong fluorescence of CNNS can be quenched by Cu²⁺via photoinduced electron transfer（PET）.When adding adenosine 5’-diphosphate（ADP）was added into the solution,the combination between ADP and Cu²⁺weakens its interaction with CNNS,leading to a slight recovery of the quenched fluorescence of CNNS.In the presence of ADK,ADP will be converted to adenosine 5’-triphosphate（ATP）and adenosine 5’-monophosphate（AMP）based on the ADK-catalyzed reverse transphosphorylation reaction（2ADP?ATP+AMP）,ATP has higher affinity interaction with Cu²⁺than ADP,which can bind more Cu²⁺and prevent the coordination between Cu²⁺and CNNS,resulting a significant fluorescence recovery.By measuring the change of fluorescence signal,ADK can be sensitively detected,with a detection limit of 0.06 U/L.The sensor is expected to provide an efficient,highly sensitive and label-free detection platform in clinical diagnostics and biomedical research that related to ADK.In the third chapter,we demonstrated that the single stranded DNA（ssDNA）can enhance the peroxidase-like activity of the CNNS,which can be used for detecting SNP.ssDNA can adsorb on the surface of CNNS to form a stable composite material.When the substrate 3,3’,5,5’-tetramethyl benzidine（TMB）with positive charge was added,it can interact with ssDNA through electrostatic attraction and aromatic ring stacking,which greatly improves the peroxidase activity of CNNS.By using DNA to enhance the catalytic oxidation activity of nanosheets,colorimetric detection of SNP based on the nanoenzyme was realized.By functionally combining the nucleic acid with the nanotechnology,this chapter developed a universal method that not only can be applied for detection of nucleic acid,but also can for analyzing the different target molecules.