Construction And Application Of Fluorescent Biosensing Platform Based On Adenosine Aptamer Labeled Nanoparticles

Adenosine as an endogenous nucleoside distributed throughout human cells,performs extremely important signaling function in the cardiovascular system,central nervous system and endocrine system.As a potential tumor biomarker,it possesses great significance in early monitoring of disease progression.Therefore,direct monitoring of adenosine fluctuations under physiological conditions would be of utility in further illustrating their function in cancer clinical diagnosis and treatment.These traditional techniques tend to require expensive instrumentation and sophisticated operation.And the foremost limitation of these methods in application of adenosine bioassay is insensitivity.Due to the low concentration level of adenosine existing in complicated biological samples,it is urgent to establish a highly selective and sensitive method for the determination of adenosine.In order to achieve the detection of trace adenosine in biological samples,a highly selective and sensitive fluorescence detection platform was constructed in this thesis.For rapid separation,adenosine aptamer 1?ABA1?was decorated with the Fe₃O₄ magnetic nanoparticles?MNPs?via covalent linkage,while adenosine aptamer2?ABA2?was attached to Ag nanoparticles?NPs?to obtain the signal label,and these two conjugates were mixed.With the advent of samples in the second step,a unit of the ABA1-modified Fe₃O₄ MNPs and ABA2-modified Ag NPs self assembled to form sandwich structure by capturing two adenosine molecules specifically.Once the sandwich structures were isolated from the mixture and re-dispersed in buffer through magnetic separation and wash processes,a certain amount of CdTe quantum dots?QDs?was added.Finally,the fluorescence intensity of the mixture solution was measured.Ag NPs in sandwich structure,which amount was in proportion to the amount of adenosine in samples,exhibit strong fluorescence quenching function on CdTe QDs,leading to the final fluorescence signal output gradually decreased with the increasing adenosine concentrations.The related experimental conditions such as concentration of Fe₃O₄MNPs-COOH,the concentration of ABA1,and the quenching time of Ag NPs to CdTe QDs were optimized.Then,the structure and morphology were characterized by Fourier infrared spectroscopy,ultraviolet spectroscopy,electrochemical methods and high resolution transmission electron microscope to confirm the successful synthesis of adenosine platform.The interference study demonstrated that the adenosine platform possessed excellent selectivity.Under the optimum conditions,the fluorescence intensity of the system linearly decreases with the increment of adenosine in the range of 0.1 nM-30 nM,with the correlation coefficient of 0.99 and limit of detection of 0.06 nM.On the basis of the successful establishment of the method,adenosine content was measured in the urine samples of three healthy volunteers using the spiked recovery test method.The results were in accordance with the normal range and were measured by comparative high performance liquid chromatography.The assay only takes 40 min which is the shortest among the aptamer-based methods ever reported.Furthermore,the platform holds great potential for monitoring various target molecules in the biomedical field and clinical diagnosis for its high efficiency and low cost.